Identification of novel factors that block programmed cell death or apoptosis by targeting JNK

ABSTRACT

Methods and compositions for modulating apoptosis by acting on the c-Jun-N-terminal kinase (JNK) pathway and assays for the isolation of agents capable of modulating apoptosis, including modulators of the JNK pathway are disclosed. A method of modulating JNK pathway independent of Gadd46β is disclosed. Methods and compositions are presented for the preparation and use of novel therapeutic compositions for modulating diseases and conditions associated with elevated or decreased apoptosis.

This application claims priority to 60/526,231, filed Dec. 2, 2003 andis a continuation-in-part of U.S. Ser. No. 10/626,905, filed Jul. 25,2003 which is a continuation-in-part of U.S. Ser. No. 10/263,330, filedOct. 2, 2002 which claims priority to 60/328,811, filed Oct. 12, 2001and 60/326,492, filed Oct. 2, 2001.

BACKGROUND

Methods and compositions that modulate apoptosis are based on blockingor stimulating components of cell survival or death pathways fromNF-κB/IκB through gene activation, to Gadd45β interacting withcomponents of the JNK pathway such as MKK7. Gadd45 β-independent JNKmodulation exists in certain cell types to regulate apoptosis or cellsurvival. The JNK pathway is a focus for control of a cell's progresstowards survival or death.

Apoptosis or programmed cell death is a physiologic process that plays acentral role in normal development and tissue homeostasis. Many factorsinteract in complex pathways to lead to cell death or cell survival.

A. NF-κB 1. NF-κB in Immune and Inflammatory Responses

NF-κB transcription factors are coordinating regulators of innate andadaptive immune responses. A characteristic of NF-κB is its rapidtranslocation from cytoplasm to nucleus in response to a large array ofextra-cellular signals, among which is tumor necrosis factor (TNFα).NF-κB dimers generally lie dormant in the cytoplasm of unstimulatedcells, retained there by inhibitory proteins known as IκBs, and can beactivated rapidly by signals that induce the sequential phosphorylationand proteolytic degradation of IκBs. Removal of the inhibitor allowsNF-κB to migrate into the cell nucleus and rapidly induce coordinatesets of defense-related genes, such as those encoding numerouscytokines, growth factors, chemokines, adhesion molecules and immunereceptors. In evolutionary terms, the association between cellulardefense genes and NF-κB dates as far back as half a billion years ago,because it is found in both vertebrates and invertebrates. While in thelatter organisms, NF-κB factors are mainly activated by Toll receptorsto induce innate defense mechanisms. In vertebrates, these factors arealso widely utilized by B and T lymphocytes to mount cellular andtumoral responses to antigens.

Evidence exists for roles of NF-κB in immune and inflammatory responses.This transcription factor also plays a role in widespread humandiseases, including autoimmune and chronic inflammatory conditions suchas asthma, rheumatoid arthritis, and inflammatory bowel disease. Indeed,the anti-inflammatory and immunosuppressive agents that are most widelyused to treat these conditions such as glucocorticoids, aspirin, andgold salts, work primarily by suppressing NF-κB.

TNFα is arguably the most potent pro-inflammatory cytokine and one ofthe strongest activators of NF-κB. In turn, NF-κB is a potent inducer ofTNFα, and this mutual regulation between the cytokine and thetranscription factor is the basis for the establishment of a positivefeedback loop, which plays a central role in the pathogenesis of septicshock and chronic inflammatory conditions such as rheumatoid arthritis(RA) and inflammatory bowel disease (IBD). Indeed, the standardtherapeutic approach in the treatment of these latter disorders consistsof the administration of high doses of NF-κB blockers such as aspirinand glucocorticoids, and the inhibition of TNFα by the use ofneutralizing antibodies represents an effective tool in the treatment ofthese conditions. However, chronic treatment with NF-κB inhibitors hasconsiderable side effects, including immunosuppressive effects, and dueto the onset of the host immune response, patients rapidly becomerefractory to the beneficial effects of anti-TNFα neutralizingantibodies.

2. NF-κB and the Control of Apoptosis

In addition to coordinating immune and inflammatory responses, theNF-κB/Rel group of transcription factors controls apoptosis. Apoptosis,that is, programmed cell death (PCD), is a physiologic process thatplays a central role in normal development and tissue homeostasis. Thehallmark of apoptosis is the active participation of the cell in its owndestruction through the execution of an intrinsic suicide program. Thekey event in this process is the activation by proteolytic cleavage ofcaspases, a family of evolutionarily conserved proteases. One pathway ofcaspase activation, or “intrinsic” pathway, is triggered by Bcl-2 familymembers such as Bax and Bak in response to developmental orenvironmental cues such as genotoxic agents. The other pathway isinitiated by the triggering of “death receptors” (DRs) such asTNF-receptor 1 (TNF-R1), Fas (CD95), and TRAIL-R1 and R2, and depends onthe ligand-induced recruitment of adaptor molecules such as TRADD andFADD to these receptors, resulting in caspase activation.

The deregulation of the delicate mechanisms that control cell death cancause serious diseases in humans, including autoimmune disorders andcancer. Indeed, disturbances of apoptosis are just as important to thepathogenesis of cancer as abnormalities in the regulation of the cellcycle. The inactivation of the physiologic apoptotic mechanism alsoallows tumor cells to escape anti-cancer treatment. This is becausechemotherapeutic agents, as well as radiation, ultimately use theapoptotic pathways to kill cancer cells.

Evidence including analyses of various knockout models—suggests thatactivation of NF-κB is required to antagonize killing cells by numerousapoptotic triggers, including TNFα and TRAIL. Indeed, most cells arecompletely refractory to TNFα cytotoxicity, unless NF-κB activation orprotein synthesis is blocked. Remarkably, the potent pro-survivaleffects of NF-κB serve a wide range of physiologic processes, includingB lymphopoiesis, B- and T-cell co stimulation, bone morphogenesis, andmitogenic responses. The anti-apoptotic function of NF-κB is alsocrucial to ontogenesis and chemo- and radio-resistance in cancer, aswell as to several other pathological conditions.

There is evidence to suggest that JNK is involved in the apoptoticresponse to TRAIL. First, the apoptotic mechanisms triggered by TRAIL-Rsare similar to those activated by TNF-R1. Second, as with TNF-R1, ligandengagement of TRAIL-Rs leads to potent activation of both JNK and NF-κB.Thirdly, killing by TRAIL is blocked by this activation of NF-κB.Nevertheless, the role of JNK in apoptosis by TRAIL has not been yetdemonstrated.

The triggering of TRAIL-Rs has received wide attention as a powerfultool for the treatment of certain cancers, and there are clinical trialsinvolving the administration of TRAIL. This is largely because, unlikenormal cells, tumor cells are highly susceptible to TRAIL-inducedkilling. The selectivity of the cytotoxic effects of TRAIL for tumorcells is due, at least in part, to the presence on normal cells ofso-called “decoy receptors”, inactive receptors that effectivelyassociate with TRAIL, thereby preventing it from binding to thesignal-transuding DRs, TRAIL-R1 and R2. Decoy receptors are insteadexpressed at low levels on most cancer cells. Moreover, unlike with FasLand TNFα, systemic administration of TRAIL induces only minor sideeffects, and overall, is well-tolerated by patients.

Cytoprotection by NF-κB involves activation of pro-survival genes.However, despite investigation, the bases for the NF-κB protectivefunction during oncogenic transformation, cancer chemotherapy, and TNFαstimulation remain poorly understood. With regard to TNF-Rs, protectionby NF-κB has been linked to the induction of Bcl-2 family members,Bcl-X_(L) and A1/Bfl-1, XIAP, and the simultaneous upregulation ofTRAF1/2 and c-IAP1/2. However, TRAF2, c-IAP1, Bcl-X_(L), and XIAP arenot significantly induced by TNFα in various cell types and are found atnear-normal levels in several NF-κB deficient cells. Moreover, Bcl-2family members, XIAP, or the combination of TRAFs and c-IAPs can onlypartly inhibit PCD in NF-κB null cells. In addition, expression of TRAF1and A1/Bfl-1 is restricted to certain tissues, and many cell typesexpress TRAF1 in the absence of TRAF2, a factor needed to recruit TRAF1to TNF-R1. Other putative NF-κB targets, including A20 and IEX-1L, areunable to protect NF-κB deficient cells or were questioned to haveanti-apoptotic activity. Hence, these genes cannot fully explain theprotective activity of NF-κB.

3. NF-κB in Oncogenesis and Cancer Therapy Resistance

NF-κB plays a role in oncogenesis. Genes encoding members of the NF-κBgroup, such as p52/p100, Rel, and RelA and the IκB-like protein Bcl-3,are frequently rearranged or amplified in human lymphomas and leukemias.Inactivating mutations of IκBα are found in Hodgkin's lymphoma (HL).NF-κB is also linked to cancer independently of mutations or chromosomaltranslocation events. Indeed, NF-κB is activated by most viral andcellular oncogene products, including HTLV-I Tax, EBV EBNA2 and LMP-1,SV40 large-T, adenovirus E1A, Bcr-Abl, Her-2/Neu, and oncogenic variantsof Ras. Although NF-κB participates in several aspects of oncogenesis,including cancer cell proliferation, the suppression of differentiation,and tumor invasiveness, direct evidence from both in vivo and in vitromodels suggests that its control of apoptosis is important to cancerdevelopment. In the early stages of cancer, NF-κB suppresses apoptosisassociated with transformation by oncogenes. For instance, uponexpression of Bcr-Abl or oncogenic variants of Ras—one of the mostfrequently mutated oncogenes in human tumors—inhibition of NF-κB leadsto an apoptotic response rather than to cellular transformation.Tumorigenesis driven by EBV is also inhibited by IκBαM—a super-activeform of the NF-κB inhibitor, IκBα. In addition, NF-κB is essential formaintaining survival of a growing list of late stage tumors, includingHL, diffuse large B cell lymphoma (DLBCL), multiple myeloma, and ahighly invasive, estrogen receptor (ER) in breast cancer. Both primarytissues and cell line models of these malignancies exhibitconstitutively high NF-κB activity. Inhibition of this aberrant activityby IκBαM or various other means induces death of these cancerous cells.In ER breast tumors, NF-κB activity is often sustained by PI-3K and Akt1kinases, activated by over-expression of Her-2/Neu receptors.Constitutive activation of this Her-2/Neu/PI-3K/Akt1/NF-κB pathway hasbeen associated with the hormone-independent growth and survival ofthese tumors, as well as with their well-known resistance to anti-cancertreatment and their poor prognosis. Due to activation of this pathwaycancer cells also become resistant to TNF-R and Fas triggering, whichhelps them to evade immune surveillance.

Indeed, even in those cancers that do not contain constitutively activeNF-κB, activation of the transcription factors by ionizing radiation orchemotherapeutic drugs (e.g. daunorubicin and etoposide) can blunt theability of cancer therapy to kill tumor cells. In fact, certain tumorscan be eliminated in mice with CPT-11 systemic treatment and adenoviraldelivery of IκBαM.

B. JNK 1. Roles of JNK in Apoptosis

The c-Jun-N-terminal kinases (JNK1/2/3) are the downstream components ofone of the three major groups of mitogen-activated protein kinase (MAPK)cascades found in mammalian cells, with the other two consisting of theextracellular signal-regulated kinases (ERK1/2) and the p38 proteinkinases (p38α/β/γ/δ). Each group of kinases is part of a three-modulecascade that include a MAPK (JNKs, ERKs, and p38s), which is activatedby phosphorylation by a MAPK kinase (MAPKK), which in turn is activatedby phosphorylation by a MAPKK kinase (MAPKKK). Whereas activation of ERKhas been primarily associated with cell growth and survival, by andlarge, activation of JNK and p38 have been linked to the induction ofapoptosis. Using many cell types, it was shown that persistentactivation of JNK induces cell death, and that the blockade of JNKactivation by dominant-negative (DN) inhibitors prevents killing by anarray of apoptotic stimuli. The role of JNK in apoptosis is alsodocumented by the analyses of mice with targeted disruptions of jnkgenes. Mouse embryonic fibroblasts (MEFs) lacking both JNK1 and JNK2 arecompletely resistant to apoptosis by various stress stimuli, includinggenotoxic agents, UV radiation, and anisomycin, and jnk3−/− neuronsexhibit a severe defect in the apoptotic response to excitotoxins.Moreover, JNK2 was shown to be required for anti-CD3-induced apoptosisin immature thymocytes.

However, while the role of JNK in stress-induced apoptosis is wellestablished, its role in killing by DRs such as TNF-R1, Fas, andTRAIL-Rs has remained elusive. Some initial studies have suggested thatJNK is not a critical mediator of DR-induced killing. This was largelybased on the observation that, during challenge with TNFα, inhibition ofJNK activation by DN mutants of MEKK1—an upstream activator of JNK hadno effect on cell survival. In support of this view, it was also notedthat despite their resistance to stress-induced apoptosis, JNK nullfibroblasts remain sensitive to killing by Fas. In contrast, anotherearly study using DN variants of the JNK kinase, MKK4/SEK1, had insteadindicated an important role for JNK in pro-apoptotic signaling by TNF-R.

2. Roles of JNK in Cancer

JNK is potently activated by several chemotherapy drugs and oncogeneproducts such as Bcr-Abl, Her-2/Neu, Src, and oncogenic Ras. Hence,cancer cells must adopt mechanisms to suppress JNK-mediated apoptosisinduced by these agents. Indeed, non-redundant components of the JNKpathway (e.g. JNKK1/MKK4) have been identified as candidate tumorsuppressors, and the well-characterized tumor suppressor BRCA1 is apotent activator of JNK and depends on JNK to induce death. Some of thebiologic functions of JNK are mediated by phosphorylation of the c-Junoncoprotein at S63 and S73, which stimulates c-Jun transcriptionalactivity. However, the effects of c-Jun on cellular transformationappear to be largely independent of its activation by JNK. Indeed,knock-in studies have shown that the JNK phospho-acceptor sites of c-Junare dispensable for transformation by oncogenes, in vitro. Likewise,some of the activities of JNK in transformation and apoptosis, as wellas in cell proliferation, are not mediated by c-Jun phosphorylation. Forinstance, while mutations of the JNK phosphorylation sites of c-Jun canrecapitulate the effects of JNK3 ablation in neuronal apoptosis—which isdependent on transcriptional events—JNK-mediated apoptosis in MEFs doesnot require new gene induction by c-Jun. Moreover, JNK also activatesJunB and JunD, which act as tumor suppressors, both in vitro and invivo. Inhibition of JNK in Ras-transformed cells is reported to have noeffect on anchorage-independent growth or tissue invasiveness. Hence,JNK and c-Jun likely have independent functions in apoptosis andoncogenesis, and JNK is not required for transformation by oncogenes insome circumstances, but may instead contribute to suppresstumorigenesis. Indeed, the inhibition of JNK might represent a mechanismby which NF-κB promotes oncogenesis and cancer chemoresistance.

C. Biologic Functions of Gadd45 Proteins

gadd45β (also known as Myd118) is one of three members of the gadd45family of inducible genes, also including gadd45α (gadd45) and gadd45γ(oig37/cr6/grp17). Gadd45 proteins are regulated primarily at thetranscriptional level and have been implicated in several biologicalfunctions, including G2/M cell cycle checkpoints and DNA repair. Thesefunctions were characterized with Gadd45α and were linked to the abilityof this factor to bind to PCNA, core histones, Cdc2 kinase, and p21.Despite sequence similarity to Gadd45α, Gadd45β exhibits somewhatdistinct biologic activities, as for instance, it does not appear toparticipate in negative growth control in most cells. Over-expression ofGadd45 proteins has also been linked to apoptosis in some systems.However, it is not clear that this is a physiologic activity, because inmany other systems induction of endogenous Gadd45 proteins is associatedwith cytoprotection, and expression of exogenous polypeptides does notinduce death. Finally, Gadd45 proteins have been shown to associate withMEKK4/MTK1 and have been proposed to be initiators of JNK and p38signaling. Other reports have concluded that expression of theseproteins does not induce JNK or p38 in various cell lines, and that theendogenous products make no contribution to the activation of thesekinases by stress. The ability of Gadd45 proteins to bind to MEKK4supports the existence of a link between these proteins and kinases inthe MAPK pathways. Studies using T cell systems, have implicated Gadd45γin the activation of both JNK and p38, and Gadd45β in the regulation ofp38 during cytokines responses.

D. Summary

Although many important cellular processes have been investigated, muchis unproven, particularly with respect to the cellular pathwaysresponsible for controlling apoptosis. For example, the manner in whichNF-κB controls apoptosis is unclear. Elucidation of the criticalpathways responsible for modulation of apoptosis is necessary in orderGadd45β in to develop new therapeutics capable of treating a variety ofdiseases that are associated with aberrant levels of apoptosis.

Inhibitors of NF-κB are used in combination with standard anti-canceragents to treat cancer patients, such as patients with HL or multiplemyeloma. Yet, therapeutic inhibitors (e.g. glucocorticoids) only achievepartial inhibition of NF-κB and exhibit considerable side effects, whichlimits their use in humans. A better therapeutic approach might be toemploy agents that block, rather than NF-κB, its downstreamanti-apoptotic effectors in cancer cells. However, despiteinvestigation, these effectors remain unknown.

SUMMARY

Gadd45β independent inactivation of JNKK2/MKK7 is disclosed. SpecificGadd45β derived peptides bind to and inactivate JNKK2/MKK7.

The JNK pathway is a focus for control of pathways leading to programmedcell death: 1) in addition to playing a role in stress-inducedapoptosis, JNK activation is necessary for efficient cell killing byTNF-R1, as well as by other DRs such as Fas and TRAIL-Rs; 2) theinhibition of the JNK cascade represents a protective mechanism by NF-κBagainst TNFα-induced cytotoxicity; 3) suppression of JNK activationmight represent a general protective mechanism by NF-κB and is likely tomediate the potent effects of NF-κB during oncogenesis and cancerchemoresistance; 4) inhibition of JNK activation and cytoprotection byNF-κB involve the transcriptional activation of gadd45β; 5) Gadd45βprotein blocks JNK signaling by binding to and inhibiting JNKK2/MKK7—aspecific and non-redundant activator of JNK. JNKK2 and MKK7 are usedinterchangably.

Gadd45β is required to block apoptosis induced by TNFα-and, at least infibroblasts, there is an additional factor binding to “peptide 2”described herein, required for this function. The Gadd45β-interactiondomains of JNKK2 and the JNKK2-binding surface of Gadd45β wereidentified. This facilitated the isolation of cell-permeable peptidesand small molecules that are able to interfere with the ability ofGadd45β, and thereby of NF-κB, to block JNK activation and preventapoptosis. The 69-86 amino acidic region of Gadd45β is sufficient tobind to MKK7 and a slightly longer region of Gadd45β (i.e. amino acids60-86) is sufficient to also inhibit MKK7 activity. This information isvery useful for modulating MKK7 activity and thereby apoptosis in vivo.Cell-permeable peptides containing this peptidic portion of Gadd45β canbe used in vivo to block TNFα-induced apoptosis in cells. This providesa means for blocking apoptosis in diseases such as neurodegenerativedisorders, stroke, myocardial infraction.

A method for modulating pathways leading to programmed cell deathincludes the steps of obtaining a peptide designated herein “peptide 2”that has an amino acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQID NO: 1) and regulating the JNK pathway by use of the peptide or by acomposition developed from knowledge of the peptide.

A method to identify factors that regulate JNK pathway leading toprogrammed cell death includes the steps of obtaining a peptide that hasan amino acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1)and identifying factors that interact with the peptide.

A cDNA molecule sufficient to encode a petide of amino acid sequenceNH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1) also regulates JNKpathway.

A method to identify agents that modulate JNK signaling includes thesteps of: (a) determining whether the agent binds to a peptide of aminoacid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); and (b)assaying for activity of the bound agent to determine the effect on JNKsignalling.

A method for screening and identifying an agent that modulates JNKactivity in vivo includes the steps of:

-   -   (a) obtaining a candidate agent that interacts with JNKK2        independent of Gadd45β;    -   (b) administering the agent to a non-human animal; and    -   (c) determining the level of JNK activity in the animal compared        to JNK activity in animals not receiving the agent.

A method for screening for a modulator of the JNK pathway includes thesteps of:

-   -   (a) obtaining a candidate modulator of the JNK pathway, wherein        the candidate modulator is capable of binding to a peptide that        has an amino acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH        (SEQ ID NO: 1);    -   (b) administering the candidate modulator to a cancer cell;    -   (c) determining the ability of the candidate modulator to        modulate the JNK pathway, including either upregulation or        downregulation of the JNK pathway and assaying the levels of up        or down regulation.

A method of treating degenerative disorders and other conditions causedby effects of apoptosis in affected cells includes the steps of:

-   -   (a) obtaining a molecule that interferes with the activation of        JNK signaling independent of Gadd45β; and    -   (b) contacting the affected cells with the molecule.

A method of aiding the immune system to kill cancer cells by augmentingJNK signaling includes the steps of:

-   -   (a) obtaining an inhibitor to block JNK signaling independent of        Gadd45β; and    -   (b) contacting the cancer cells with the inhibitor.

The molecule interferes with the activation of JNKK2 independent ofGadd45β.

A method of identifying JNKK2-interacting factors includes the steps of:

-   -   (a) providing a peptide comprising an amino acid sequence        TGHVIAVKQMRRSGNKEENKRILMD (SEQ ID NO: 1) as a bait; and    -   (b) identifying factors that interact with the peptide.

A method to determine agents that interfere with binding of JNKK2 to amolecule capable of binding to positions 142-166(TGHVIAVKQMRRSGNKEENKRILMD SEQ ID NO: 1) of the full length JNKK2includes the steps of:

-   -   (a) obtaining an agent that interferes with the binding of the        molecule to positions 142-166 (TGHVIAVKQMRRSGNKEENKRILMD SEQ ID        NO: 1) of the full length JNKK2;    -   (b) contacting a cell with the agent under conditions that would        induce transient JNK activation; and    -   (c) comparing cells contacted with the agent to cells not        contacted with the agent to determine if the JNK pathway is        activated.

A method for obtaining a mimetic that is sufficient to suppress JNKactivation by interacting with JNKK2, includes the steps of: (a)designing the mimetic to mimic the function of peptideNH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1); (b) contacting themimetic to a system that comprises the JNK pathway; and (c) determiningwhether there is suppression of JNK signalling.

A method of treating TNFα and NF-□B-dependent disorders, includes thesteps of:

-   -   (a) obtaining a molecule that interferes with the activation of        JNK signaling independent of Gadd45f3; and    -   (b) contacting the affected cells with the molecule.

The disorder may be rheumatoid arthritis, inflammatory bowel disease,and cancer.

A peptide comprising an amino acid sequence TGHVIAVKQMRRSGNKEENKRILMD(SEQ ID NO: 1), can be used to treat TNFα and NF-□B dependent disorders.

A peptide mimetic that resembles a peptide comprising an amino acidsequence TGHVIAVKQMRRSGNKEENKRILMD (SEQ ID NO: 1), can be used to treatTNFα and NF-□B depend disorders

An inhibitor of the cellular factor that binds to a peptide comprisingan amino acid sequence TGHVIAVKQMRRSGNKEENKRILMD (SEQ ID NO: 1), can beused to treat TNFα and NF-□B dependent disorders.

A method of aiding the host immune system to kill cancer cells byaugmenting JNK signaling, includes the steps of:

-   -   (c) obtaining an inhibitor that blocks JNK signaling independent        of Gadd45f3; and    -   (d) contacting the cancer cells with the inhibitor.

A method of identifying JNKK2-interacting cellular factors, the methodcomprising:

-   -   (e) providing a peptide comprising an amino acid sequence        TGHVIAVKQMRRSGNKEENKRILMD (SEQ ID NO: 1); and    -   (f) identifying cellular factors that interact with the peptide.

A peptide molecule comprising an amino acid sequenceTGHVIAVKQMRRSGNKEENKRILMD (SEQ ID NO: 1).

A pharmaceutical composition includes a peptide comprising an amino acidsequence TGHVIAVKQMRRSGNKEENKRILMD (SEQ ID NO: 1) and a pharmaceuticallyacceptable carrier. The peptide is cell permeable.

A peptide consisting essentially of a contiguous amino acid sequenceidentical to the amino acid sequence of Gadd45β, selected from the groupconsisting of peptide whose amino acid sequences are from positions60-86 (AIDEEEEDDIALQIHFTLIQSFCCDND SEQ ID NO: 2) and 69-86(IALQIHFTLIQSFCCDND SEQ ID NO: 3), the molecule capable of binding toMKK7.

A cell permeable peptide includes an amino acid sequence functionallyequivalent to that of positions 60-86 of Gadd45β protein.

A method to block apoptosis, the method includes the step of:

-   -   (g) obtaining a peptide whose amino acid sequence is selected        from the group consisting of peptides whose amino acid sequences        are from positions 60-86 (AIDEEEEDDIALQIHFTLIQSFCCDND SEQ ID        NO: 2) and 69-86 (IALQIHFTLIQSFCCDND SEQ ID NO: 3) of Gadd45β;        and    -   (h) administering the peptide to block apoptosis by selective        inactivation of JNKK2.

The apoptosis is blocked in inflammatory diseases, neurodegenerativedisorders, stroke, and myocardial infarction.

A peptide mimetic that resembles amino acid sequences that are frompositions 60-86 (AIDEEEEDDIALQIHFTLIQSFCCDND SEQ ID NO: 2) and 69-86(IALQIHFTLIQSFCCDND SEQ ID NO: 3) of Gadd45β, can be used to blockapoptosis.

A method to identify inhibitors of Gadd45β, includes the steps of:

-   -   (a) screening for a candidate compound that interacts with        peptidic regions comprising amino acid sequences from positions        60-86 (AIDEEEEDDIALQIHFTLIQSFCCDND SEQ ID NO: 2) and 69-86        (IALQIHFTLIQSFCCDND SEQ ID NO: 3) of Gadd45β; and    -   (b) determining the ability of the candidate to bind to Gadd45β.

A cDNA molecule may encode a fragment of Gadd45 protein that issufficient to suppress JNK signaling, a peptide that corresponds toamino acids 69-113 of Gadd45β. A peptide including the amino andsequence from 69-86 is sufficient to bind to MKK7 and a slightly longerregion (amino acids 60-86) is sufficient to also inhibit MKK7activation. Cell permeable peptides including this fragment of Gadd45βcan block TNFα in vivo, to block TNFα induced apoptosis in cells.

A method for modulating pathways leading to programmed cell deathincludes the steps of obtaining a peptide that has an amino acidsequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1) andregulating the JNK pathway by use of the peptide or a compositiondeveloped from knowledge of the amino acid sequence of the peptide.

Compositions of this invention include a molecule including a bindingregion of JNKK2 characterized by the amino acid sequence from positions142-166 (TGHVIAVKQMRRSGNKEENKRILMD SEQ ID NO: 1) of the full lengthJNKK2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows Gadd45β antagonizes TNFR-induced apoptosis in NF-κB nullcells. FIG. 1A: Gadd45β as well as Gadd45α and Gadd45γ (left) rescueRelA−/− MEFs, TNFα-induced killing. Plasmids were used as indicated.Cells were treated with CHS (0.1 ug/ml or CHX plus TNFα (100 units/ml)and harvested at the indicated time points. Each column represents thepercentage of GHP+ live cells in TNFα treated cultures relative to thecultures treated with CHX alone. Values are the means of threeindependent experiments. The Figure indicates that Gadd45α, Gadd45β andGadd45γ have anti-apoptotic activity against TNFα. FIG. 1B: NF-κB null3DO cells are sensitive to TNFα Cell lines harboring IκBαM or neoplasmids were treated with TNFα (300 units/ml) and harvested at 14hours. Columns depict percentages of live cells as determined by PIstaining. Western blots show levels of IκβαM protein (bottom panels).FIG. 1C: 3DO IκβαM-Gadd45β cells are protected from TNFα killing. Cellsare indicated. Cells were treated with TNFα (25 units/ml) or leftuntreated and harvested at the indicated time points. Each valuerepresents the mean of three independent experiments and expresses thepercentages of live cells in treated cultures relatively to controls(left). PI staining profiles of representative clones after an 8-hourincubation with or without TNFα (right panel, TNFα and US.respectively). FIG. 1D: Protection correlates with levels of Gadd45β ofthe 8-hr. time point experiment shown in (C) with the addition of twoIκB-Gadd4513 lines. Western blots are as indicated (lower panels),. FIG.1E: Gadd45β functions downstream of NF-κB complexes. EMSA with extractsof untreated and TNFα-treated 3DO cells. Composition of the κB-bindingcomplexes was assessed by using supershifting antibodies. FIG. 1F showsGadd45β is essential to antagonize TNFα-induced apoptosis. 3DO linesharboring anti-sense Gadd45β (AS-Gadd45β) or empty (Hygro) plasmids weretreated with CHX (0.1 μg/ml) plus or minus TNFα (1000 units/ml) andanalyzed at 14 hours by nuclear PI staining. Low concentration of CHXwas used to lower the threshold of apoptosis. Each column valuerepresents the mean of three independent experiments and was calculatedas described in FIG. 1C.

FIGS. 2 a-2 d shows Gadd45β is a transcriptional target of NF-κB. FIG. 2a: Northern blots with RNA from untreated and TNFα (1000 u/ml) treatedRelA−/− and +/+ MEF. Probes are as indicated. FIG. 2 b-2 d: 3 DO IκβαMcells and controls were treated with TNFα (1000 u/ml). PMA (50 g/ml)plus ionomycin (1 μM) or daunorubicin (0.5 μM), respectively andanalyzed as in FIG. 2 a.

FIGS. 3A-3E shows Gadd45β prevents caspase activation in NF-κB nullcells. FIG. 3A:Gadd45-dependent blockade of caspase activity. 3DO lineswere treated with TNFα (50 units/ml) and harvested at the indicated timepoints for the measurement of caspase activity by in vitro fluorometricassay. Values express fluorescence units obtained after subtracting thebackground. FIG. 3B: Gadd45α inhibits TNFα-induced processing of Bid andpro-caspases. Cell were treated as described in FIG. 2A. Closed and openarrowheads indicate unprocessed and processed proteins, respectively.FIG. 3C: Gadd45, completely abrogates TNFα-induced mitochondrialdepolarization in NFκB-null cells. 3DO lines and the TNFα treatment wereas described in FIGS. 3A and B. Each value represents the mean of threeindependent experiments and expresses the percentage of JC-1⁺ cells ineach culture. FIG. 3D-#: Gadd45β inhibits cisplatinum- anddaunorubicin-induced toxicity. Independently generated IκBαM-Gadd45β and-Hygro clones were treated for 24 hr with (concentration) 0.025 μMcisplatinum (FIG. 3D) or with 0.025 μM daunorubicin (FIG. 3E) asindicated. Values represent percentages of live cells as assessed bynuclear PI staining and were calculated as described in FIG. 1C.

FIG. 4 shows Gadd45β is a physiologic inhibitor of JNK signaling. FIG. 4a: Western blots showing kinetics of JNK activation by TNFα (1000 U/ml)in IκBαM-Hygro and IκBαM-Gadd45β 3DO clones. Similar results wereobtained with four additional IκBαM—Gadd45β and three IκBαM—Hygroclones. FIG. 4 b: Western blots showing ERK, p38, and JNKphosphorylation in 3DO clones treated with TNFα for 5 minutes. FIG. 4 d:Western blots (top and middle) and kinase assays (bottom) showing JNKactivation in anti-sense-Gadd45β and Hygro clones treated with TNFα asin (a). FIG. 4 c: JNK activation by hydrogen peroxide (H₂O₂, 600 μM) andsorbitol (0.3M) in IκBαM-Hygro and IκBαM-Gadd45β clones. Treatments werefor 30 minutes.

FIG. 5 a-e shows the inhibition of JNK represents a protective mechanismby NF-κB. FIG. 5 a: Kinetics of JNK activation by TNKα (1000 U/ml) in3DO—IκBαM and 3DO-Neo clones. Western blots with antibodies specific forphosphorylated (P) or total JNK (top and middle, respectively) and JNKkinase assays (bottom). Similar results were obtained with twoadditional IκBαM and five Neo clones. FIG. 5 b: Western blots (top andmiddle) and kinase assays (bottom) showing JNK activation in RelA−/− and+/+ MEFs treated as in (a). FIG. 5 c: Western blots (top and middle) andkinase assays (bottom) showing JNK activation in parental 3DO cellstreated with TNFα (1000 U/ml), TNFα plus CHX (10 μg/ml), or CHX alone.CHX treatments were carried out for 30 minutes in addition to theindicated time. FIG. 5 d: Survival of transfected RelA−/− MEFs followingtreatment with TNFα (1000 U/ml) plus CHX (0.1 g/ml) for 10 hours.Plasmids were transfected as indicated along with pEGFP (Clontech). FIG.5 e: Survival of 3DO-IκBαM cells pretreated with MAPK inhibitors for 30minutes and then incubated with either TNFα (25 U/ml) or PBS for anadditional 12 hours. Inhibitors (Calbiochem) and concentrations are asindicated. In (d) and (e), values represent the mean of threeindependent experiments.

FIG. 6 shows gadd45β expression is strongly induced by RelA, but not byRel or p50. Northern blots showing expression of gadd45β transcripts inHtTA-1 cells and HtTA-p50, HtTA-p50, HtTA-RelA, and HtTA-CCR43 cellclones maintained in the presence (0 hours) or absence of tetracyclinefor the times shown. Cell lines, times after tetracycline withdrawal,and ³²P-labeled probes specific to gadd45β, ikbα, relA, p50, rel, orcontrol gapdh cDNAs, are as indicated. The tetracycline-inducible nf-kbtransgenes are boxed. Transcripts from the endogenous p105 gene and p50transgene are indicated.

FIG. 7 shows gadd45β expression correlates with NF-κB activity in B celllines. Northern blots showing constitutive and inducible expression ofgadd45β in 70Z/3 pre-B cells and WEHI-231 B cells (lanes 1-5 and 5-5,respectively). Cells were either left untreated (lanes 1, 6, and 11) ortreated with LPS (40 μg/ml) or PMA (100 ng/ml) and harvested for RNApreparation at the indicated time points. Shown are two differentexposures of blots hybridized with a ³²P-labeled probe specific to themouse gadd45β cDNA (top panel, short exposure; middle panel, longexposure). As a loading control, blots were re-probed with gapdh (bottompanel).

FIG. 8 shows the sequence of the proximal region of the murine gadd45βpromoter (SEQ ID NO: 35). Strong matches for transcription factorbinding sites are underlined and cognate DNA-binding factors areindicated. Positions where murine and human sequences are identical,within DNA stretches of high homology, are highlighted in gray. Withinthese stretches, gaps introduced for alignment are marked with dashes.κB binding sites that are conserved in the human promoter are boxed. Apreviously identified transcription start site is indicated by anasterisk, and transcribed nucleotides are italicized. Numbers on theleft indicate the base pair position relative to the transcription startsite. It also shows the sequence of the proximal region of the murinegadd45β promoter. To understand the regulation of Gadd45β by NF-□B, themurine gadd45β promoter was cloned. A BAC library clone containing thegadd45β gene was isolated, digested with Xhol, and subcloned into pBS.The 7384 b XhoI fragment containing gadd45β was completely sequenced(accession number: AF441860), and portions were found to match sequencespreviously deposited in GeneBank (accession numbers: AC073816, AC073701,and AC091518). This fragment harbored the genomic DNA region spanningfrom ˜5.4 kb upstream of a previously identified transcription startsite to near the end of the fourth exon of gadd45β. A TATA box waslocated at position −56 to −60 relative to the transcription start site.The gadd45β promoter also exhibited several NF-□B-binding elements.Three strong □B sites were found in the proximal promoter region atpositions −377/−368, −426/−417, and −447/−438; whereas a weaker site waslocated at position −1159/−1150 and four other matches mapped furtherupstream at positions −2751/−2742, −4525/−4516, −4890/−4881, and−5251/−5242 (gene bank accession number AF441860). Three □B consensussites within the first exon of gadd45β (+27/+36, +71/+80, and+171/+180). The promoter also contained a Sp1 motif (−890/−881) andseveral putative binding sites for other transcription factors,including heat shock factor (HSF) 1 and 2, Ets, Stat, AP1, N-Myc, MyoD,CREB, and C/EBP.

To identify conserved regulatory elements, the 5.4 kb murine DNAsequence located immediately upstream of the gadd45β transcription startsite was aligned with the corresponding human sequence, previouslydeposited by the Joint Genome Initiative (accession number: AC005624).The −1477/−1197 and −466/−300 regions of murine gadd45β were highlysimilar to portions of the human promoter, suggesting that these regionscontain important regulatory elements (highlighted in gray are identicalnucleotides within regions of high homology). A less well-conservedregion was identified downstream of position −183 to the beginning ofthe first intron. Additional shorter stretches of homology were alsoidentified. No significant similarity was found upstream of position−2285. The homology region at −466/−300 contained three κB sites(referred to as κB-1, κB-2, and κB-3), which unlike the other κB sitespresent throughout the gadd45β promoter, were conserved among the twospecies. These findings suggest that these κB sites may play animportant role in the regulation of gadd45β, perhaps accounting for theinduction of gadd45β by NF-κB.

FIG. 9 shows the murine gadd45β promoter is strongly transactivated byRelA. (A) Schematic representation of CAT reporter gene constructsdriven by various portions of the murine gadd45β promoter. Numbersindicate the nucleotide position at the ends of the promoter fragmentcontained in each CAT construct. The conserved κB-1, κB-2, and κB-3sites are shown as empty boxes, whereas the TATA box and the CAT codingsequence are depicted as filled and gray boxes, whereas the TATA box andthe CAT coding sequence are depicted as filled and gray boxes,respectively. (B) Rel-A-dependent transactivation of the gadd45βpromoter. NTera-2 cells were cotransfected with individual gadd45β-CATreporter plasmids (6 μg) alone or together with 0.3, 1, or 3 μg ofPmt2t-RelA, as indicated. Shown in the absolute CAT activity detected ineach cellular extract and expressed as counts per minute (c.p.m.). Eachcolumn represents the mean of three independent experiments afternormalization to the protein concentration of the cellular extracts. Thetotal amount of transfected DNA was kept constant throughout by addingappropriate amounts of insert-less pMT2T. Each reporter constructtransfected into Ntera-2 cells with comparable efficiency, as determinedby the cotransfection of 1 μg of pEGFP (encoding green fluorescentprotein; GFP; Contech), and flow cytometric analysis aimed to assesspercentages of GFP⁺ cells and GFP expression levels.

FIG. 10 shows the gadd45β promoter contains three functional κBelements. (A) Schematic representation of wild-type and mutated−592/+23-gadd45β-CAT reporter constructs. The κB-1, κB-2, and κB-3binding sites, the TATA box, and the CAT gene are indicated as in FIG.9A. Mutated κB sites are crossed. (B) κB-1, κB-2, and κB-3 are eachrequired for the efficient transactivation of the gadd45β promoter byRelA. Ntera-2 cells were cotransfected with wild-type or mutated−592/+23-gadd45β-CAT reporter constructs alone or together with 0.3, 1,or 3 μg pMT2T-RelA, as indicated. Shown is the relative CAT activity(fold induction) over the activity observed with transfection of thereporter plasmid alone. Each column represents the mean of threeindependent experiments after normalization to the protein concentrationof the cellular extracts. Empty pMT2T vectors were used to keep theamount of transfected DNA constant throughout. pEGFP was used to controlthe transfection efficiencies of CAT plasmids, as described in FIG. 9B.

FIG. 11 shows κB elements from the gadd45β promoter are sufficient forRelA-dependent transactivation. Ntera cells were cotransfected withΔ56-κB-1/2-CAT, Δ56-κB-3-CAT, or Δ56-κB-M-CAT reporter constructs aloneor together with 0.3 or 1 μg of RelA expression plasmids, as indicated.As in FIG. 10B, columns show the relative CAT activity (fold induction)observed after normalization to the protein concentration of thecellular extracts and represent the mean of three independentexperiments. Insert-less pMT2T plasmids were used to adjust for totalamount of transfected DNA.

FIG. 12 shows gadd45β promoter κB sites bind to NF-κB complexes invitro. (A) EMSA showing binding of p/50p5 and p50/RelA complexes toκB-1, κB-2, and κB-3 (lanes 9-12, 5-8, and 1-4, respectively). Wholecell extracts were prepared from NTera-2 cells transfected withpMT2T-p50 (9 μ; lanes 1-3,5-7, and 11-12) or pMT2T-p50 (3 kg) pluspMT2T-RelA (6 μg; lanes 4, 8, and 12). Various amounts of cell extracts(0.1 μl, lanes 3, 7, and 11; 0.3 μl, lanes 2, 6, and 10; or 1 μl, lanes1, 4, 5, 8, 9, and 12) were incubated in vitro with ³²P-labeled κB-1,κB-2, or κB-3 probes, as indicated, and the protein-DNA complexes wereseparated by EMSA. NF-κB-DNA binding complexes are indicated. (B)Supershift analysis of DNA-binding NF-κB complexes. κB sites wereincubated with 1 μl of the same extracts used in (A) or of extracts fromNTera-2 cells transfected with insert-less pMT2T (lanes 1-3, 10-12, and19-21). Samples were loaded into gels either directly or afterpreincubation with antibodies directed against human p50 or RelA, asindicated. Transfected plasmids and antibodies were as shown.DNA-binding NF-κB complexes, supershifted complexes, and non-specific(n.s.) bands are labeled. (C) shows gadd45β κB sites bind to endogenousNF-κB complexes in vitro. To determine whether gadd45β-κB elements canbind to endogenous NF-κB complexes, whole cell extracts were obtainedfrom untreated and lypopolysaccharide (LPS)-treated WEHI-231 cells.Cells were treated with 40 μg/ml LPS (Escherichia coli serotype 0111:B4)for 2 hours, and 2 μl of whole cell extracts were incubated, in vitro,with ³²P-labeled gadd45β-κB probes. Probes, antibodies againstindividual NF-κB subunits, predominant DNA-binding complexes,supershifted complexes, and non-specific (n.s.) bands are as labeled.All three gadd45β-κB sites bound to both constitutively active andLPS-induced NF-κB complexes (lanes 1-3,9-11, and 17-19). κB-3 boundavidly to a slowly-migrating NF-κB complex, which was supershifted onlyby the anti-Rel antibody (lanes 4-8). This antibody also retarded themigration of the slower dimers binding to κB-2 and, much more loosely,to κB-1, but had no effect on the faster-migrating complex detected withthese probes (lanes 15 and 23, respectively). The slower complexinteracting with κB-1 and κB-2 also contained large amounts of p50 andsmaller quantities of p52 and RelA (lanes 12-14 and 20-22, RelA wasbarely detectable with κB-1). The faster complex was instead almostcompletely supershifted by the anti-p50 antibody (lanes 12 and 20), andthe residual DNA-binding activity reacted with the anti-p52 antibody(lanes 13 and 21; bottom band). With each probe, RelB dimers contributedto the κB-binding activity only marginally. Specificity of theDNA-binding complexes was confirmed by competitive binding reactionsusing unlabeled competitor oligonucleotides. Thus, the faster complexbinding to κB-1 and κB-2 was predominantly composed of p50 homodimersand contained significant amounts of p52/p52 dimers, whereas the slowerone was made up of p50/Rel heterodimers and, to a lesser extent,p52/Rel, Rel/Rel, and RelA-containing dimers. Conversely, κB-3 onlybound to Rel homodimers. Consistent with observations made withtransfected NTera-2 cells, κB-1 exhibited a clear preference for p50 andp52 homodimers, while κB-2 preferentially bound to Rel- andRelA-containing complexes. Overall, κB-3 yielded the strongestNF-κB-specific signal, whereas κB-1 yielded the weakest one.Interestingly, the in vitro binding properties of the DNA probes did notseem to reflect the relative importance of individual κB sites topromoter transactivation in vivo. Nevertheless, the findings dodemonstrate that each of the functionally relevant κB elements of thegadd45β promoter can bind to NF-κB complexes, thereby providing thebasis for the dependence of gadd45β expression on NF-κB.

FIG. 13 shows Gadd45β expression protects BJAB cells against Fas- andTRAIL-R-induced apoptosis. To determine whether Gadd45β activityextended to DRs other than TNF-Rs, stable HA-Gadd45β and Neo controlclones were generated in BJAB B cell lymphomas, which are highlysensitive to killing by both Fas and TRAIL-Rs. As shown by propidiumiodide (PI) staining assays, unlike Neo clones, BJAB clones expressingGadd45β were dramatically protected against apoptosis induced either (B)by agonistic anti-Fas antibodies (APO-1; 1 μg/ml, 16 hours) or (A) byrecombinant (r)TRAIL (100 ng/ml, 16 hours). In each case, cell survivalcorrelated with high levels of HA-Gadd45β proteins, as shown by Westernblots with anti-HA antibodies (bottom panels). Interestingly, with Fas,protection by Gadd45β was nearly complete, even at 24 hours.

FIG. 14 shows the inhibition of JNK activation protects BJAB cells fromFas induced apoptosis. Parental BJAB cells were treated for 16 hourswith anti-APO1 antibodies (1 μg/ml), in the presence or absence ofincreasing concentrations of the specific JNK blocker SP600125(Calbiochem), and apoptosis was monitored by PI staining assays. WhileBJAB cells were highly sensitive to apoptosis induced by Fas triggering,the suppression of JNK activation dramatically rescued these cells fromdeath, and the extent of cytoprotection correlated with theconcentration of SP600125. The data indicate that, unlike what waspreviously reported with MEFs (i.e. with ASK1- and JNK-deficient MEFs),in B cell lymphomas, and perhaps in other cells, JNK signaling plays apivotal role in the apoptotic response to Fas ligation. This isconsistent with findings that, in these cells, killing by Fas is alsoblocked by expression of Gadd45β (FIG. 13B). Thus, JNK might be requiredfor Fas-induced apoptosis in type 2 cells (such as BJAB cells), whichunlike type 1 cells (e.g. MEFs), require mitochondrial amplification ofthe apoptotic signal to activate caspases.

FIG. 15 shows JNK is required for efficient killing by TNFα. In FIGS. 5d and 5 e,the inhibition of JNK by either expression of DN-MKK7 or highdoses of the pharmacological blocker SB202190 rescues NF-κB null cellsfrom TNFα-induced killing. Together with the data shown in FIG. 5 a-c,these findings indicate that the inhibition of the JNK cascaderepresents a protective mechanism by NF-κB. They also suggest that theJNK cascade plays an important role in the apoptotic response to thecytokine. Thus, to directly link JNK activation to killing by TNF-R1,the sensitivity of JNK1 and JNK2 was tested in double knockoutfibroblasts to apoptosis by TNFα. Indeed, as shown in FIG. 15A, mutantcells were dramatically protected against combined cytotoxic treatmentwith TNFα (1,000 U/ml) and CHX (filled columns) for 18 hours, whereaswild-type fibroblasts remained susceptible to this treatment (emptycolumns). JNK kinase assays confirmed the inability of knockout cells toactivate JNK following TNFα stimulation (left panels). The defect in theapoptotic response of JNK null cells to TNFα plus CHX was not adevelopmental defect, because cytokine sensitivity was promptly restoredby viral transduction of MIGR1-JNKK2-JNK1, expressing constitutivelyactive JNK1 (FIG. 15B; see also left panel, JNK kinase assays). Thus,together with the data shown in FIG. 5 a-e, these latter findings withJNK null cells indicate that JNK (but not p38 or ERK) is essential forPCD by TNF-R, and confirm that a mechanism by which NF-κB protects cellsis the down-regulation of the JNK cascade by means of Gadd45β.

FIG. 16 shows Gadd45β is a potential effector of NF-κB functions inoncogenesis. Constitutive NF-κB activation is crucial to maintainviability of certain late stage tumors such as ER⁻ breast tumors.Remarkably, as shown by Northern blots, gadd45β was expressed atconstitutively high levels in ER⁻ breast cancer cell lines—which dependon NF-κB for their survival—but not in control lines or in lessinvasive, ER⁺ breast cancer cells. Of interest, in these cells, gadd45βexpression correlated with NF-κB activity. Hence, as with the control ofTNFα-induced apoptosis, the induction of gadd45β likely represents amechanism by which NF-κB promotes cancer cell survival, and therebyoncogenesis. Thus, Gadd45β is a novel target for anti-cancer therapy.

FIG. 17 shows the suppression of JNK represents a mechanism by whichNF-κB promotes oncogenesis. The ER⁻ breast cancer cell lines, BT-20 andMDA-MD-231, are well-characterized model systems of NF-κB-dependenttumorigenesis, as these lines contain constitutively nuclear NF-κBactivity and depend on this activity for their survival. In these cellsthe inhibition of NF-κB activity by well-characterized pharmacologicalblockers such as prostaglandin A1 (PGA1, 100 μM), CAPE (50 μg/ml), orparthenolide (2.5 μg/ml) induced apoptosis rapidly, as judged by lightmicroscopy. All NF-κB blockers were purchased from Biomol andconcentrations were as indicated. Treatments were carried out for 20(PGA1), 4 (parthenolide), or 17 hours (CAPE). Apoptosis was scoredmorphologically and is graphically represented as follows: ++++, 76-100%live cells; +++, 51-75% live cells; ++, 26-50% live cells; +, 1-25% livecells; −, 0% live cells. Remarkably, concomitant treatment with the JNKinhibitor SP600125 dramatically rescued breast tumor cells from thecytotoxicity induced by the inhibition of NF-κB, indicating that thesuppression of JNK by NF-κB plays an important role in oncogenesis.

FIG. 18 is a schematic representation of TNF-R1-induced pathwaysmodulating apoptosis. The blocking of the NF-κB-dependent pathway byeither a RelA knockout mutation, expression of IκBαM proteins oranti-sense gadd45β plasmids, or treatment with CHX leads to sustainedJNK activation and apoptosis. Conversely, the blocking of TNFα-inducedJNK activation by either JNK or ASK1 null mutations, expression ofDN-MKK7 proteins, or treatment with well characterized pharmacologicalblockers promotes cell survival, even in the absence of NF-κB. Theblocking of the JNK cascade by NF-RB involves the transcriptionalactivation of gadd45β. Gadd45β blocks this cascade by direct binding toand inhibition of MKK7/JNKK2, a specific and non-redundant activator ofJNK. Thus, MKK7 and its physiologic inhibitor Gadd45β, are crucialmolecular targets for modulating JNK activation, and consequentlyapoptosis.

FIG. 19 shows physical interaction between Gadd45β and kinases in theJNK pathway, in vivo. Gadd45β associates with MEKK4. However, becausethis MAPKKK is not activated by DRs, no further examination was made ofthe functional consequences of this interaction. Thus, to begin toinvestigate the mechanisms by which Gadd45β blunts JNK activation byTNF-R, the ability of Gadd45, to physically interact with additionalkinases in the JNK pathway was examined, focusing on those MAPKKKs,MAPKKs, and MAPKs that had been previously reported to be induced byTNF-Rs. HA-tagged kinases were transiently expressed in 293 cells, inthe presence or absence of FLAG-Gadd45β, and cell lysates were analyzedby co-immunoprecipitation (EP) with anti-FLAG antibody-coated beadsfollowed by Western blot with anti-HA antibodies. These assays confirmedthe ability of Gadd45β to bind to MEKK4. These co-IP assays demonstratedthat Gadd45β can also associate with ASK1, but not with otherTRAF2-interacting MAPKKKs such as MEKK1, GCK, and GCKR, or additionalMAPKKKs that were tested (e.g. MEKK3). Notably, Gadd45β also interactedwith JNKK21MKK7, but not with the other JNK kinase, JNKK1/MKK4, or withany of the other MAPKKs and MAPKs under examination, including the twop38-specific activators MKK3b and MKK6, and the ERK kinase MEK1. Similarfindings were obtained using anti-HA antibodies for IPs and anti-FLAGantibodies for Western blots. Indeed, the ability to bind to JNKK2, thedominant JNK kinase induced by TNF-R, as well as to ASK1, a kinaserequired for sustained JNK activation and apoptosis by TNFα, mayrepresent the basis for the control of JNK signaling by Gadd45β. Theinteraction with JNKK2 might also explain the specificity of theinhibitory effects of Gadd45β on the JNK pathway.

FIG. 20 shows physical interaction between Gadd45β and kinases in theJNK pathway, in vitro. To confirm the above interactions, in vitro, GSTpull-down experiments were performed. pBluescript (pBS) plasmidsencoding full-length (FL) human ASK1, MEKK4, JNKK1, and JNKK2, orpolypeptides derived from the amino- or carboxy-terminal portions ofASK1 (i.e. N-ASK1, spanning from amino acids 1 to 756, and C-ASK1,spanning from amino acids 648 to 1375) were transcribed and translatedin vitro using the TNT coupled retyculocyte lysate system (Promega) inthe presence of ³⁵S-methionine. 5 μl of each translation mix wereincubated, in vitro, with sepharose-4B beads that had been coated witheither purified glutathione-S-transferase (GST) polypeptides orGST-Gadd45β proteins. The latter proteins contained FL murine Gadd45βdirectly fused to GST. Binding assays were performed according tostandard procedures, and ³⁵S-labeled proteins that bound to beads, aswell as 2 μl of each in vitro translation mix (input), were thenresolved by SDS polyacrylamide gel electrophoresis. Asterisks indicatethe intact translated products. As shown in FIG. 20, FL-JNKK2 stronglyassociated with GST-Gadd45β, but not with GST, indicating that JNKK2 andGadd45β also interacted in vitro, and that their interaction wasspecific. Additional experiments using recombinant JNKK2 and Gadd45βhave demonstrated that this interaction is mediated by directprotein-protein contact. Consistent with in vivo findings, GST-Gadd45βalso associated with ASK1, N-ASK1, C-ASK1, and MEKK4—albeit less avidlythan with JNKK2—and weakly with JNKK1. Thus, GST pull-down experimentsconfirmed the strong interaction between Gadd45β and JNKK2 observed invivo, as well as the weaker interactions of Gadd45β with other kinasesin the JNK pathway. These assays also uncovered a weak associationbetween Gadd45β and JNKK1.

FIG. 21 shows Gadd45β inhibits JNKK2 activity in vitro. Next, thefunctional consequences, in vitro, of the physical interactions ofGadd45β with kinases in the JNK pathway were assessed. Murine and human,full-length Gadd45J3 proteins were purified from E. coli as GST-Gadd45βand His₆-tagged Gadd45β (His₆ disclosed as SEQ ID NO: 46), respectively,according to standard procedures. Prior to employing these proteins inin vitro assays, purity of all recombinant polypeptides was assuredby >98%, by performing Coomassie blue staining of SDS polyacrylamidegels. Then, the ability of these proteins, as well as of control GST andHis₆-EF3 (His₆ disclosed as SEQ ID NO: 46)proteins, to inhibit kinasesin the JNK pathways was monitored in vitro. FLAG-tagged JNKK2, JNKK1,MKK3, and ASK1 were immunoprecipitated from transiently transfected 293cells using anti-FLAG antibodies and pre-incubated for 10 minutes withincreasing concentrations of recombinant proteins, prior to the additionof specific kinase substrates (i.e. GST-JNK1 with JNKK1 and JNKK2;GST-p38□ with MKK3; GST-JNNK1 or GST-JNKK2 with ASK1). Remarkably, bothGST-Gadd45β and His₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46)effectively suppressed JNKK2 activity, in vitro, even at the lowestconcentrations that were tested, whereas control polypeptides had noeffect on kinase activity (FIG. 21A). In the presence of the highestconcentrations of Gadd45β proteins, JNKK2 activity was virtuallycompletely blocked. These findings indicate that, upon binding toGadd45β, JNKK2 is effectively inactivated. Conversely, neitherGST-Gadd45β nor His₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46) hadsignificant effects on the ability of the other kinases (i.e. JNKK1,MKK3, and ASK1) to phosphorylate their physiologic substrates, in vitro,indicating that Gadd45β is a specific inhibitor of JNKK2. Gadd45β alsoinhibited JNKK2 auto-phosphorylation.

FIG. 22A-B shows Gadd45, inhibits JNKK2 activity in vivo. The ability ofGadd45β to inhibit JNKK2 was confirmed in vivo, in 3DO cells. In thesecells, over-expression of Gadd45β blocks JNK activation by variousstimuli, and the blocking of this activation is specific, because Gadd45does not affect either the p38 or the ERK pathway. These findingssuggest that Gadd45β inhibits JNK signaling downstream of the MAPKKKmodule.

Kinase assays were performed according to procedures known to those ofskill in the art using extracts from unstimulated and TNFα-stimulated3DO cells, commercial antibodies that specifically recognize endogenouskinases, and GST-JNK1 (with JNKK2) or myelin basic protein (MBP; withASK1) substrates (FIG. 22A). Activity of JNKK1 and MKK3/6 was insteadassayed by using antibodies directed against phosphorylated (P) JNKK1 orMKK3/6 (FIG. 22B)—the active forms of these kinases. In agreement withthe in vitro data, these assays demonstrated that, in 3DO cells, Gadd45βexpression is able to completely block JNKK2 activation by TNFα (FIG.22A). This expression also partly suppressed JNKK1 activation, but didnot have significant inhibitory effects on MKK3/6—the specificactivators of p38—or ASK1 (FIG. 22A-B).

Hence, Gadd45β is a potent blocker of JNKK2—a specific activator of JNKand an essential component of the TNF-R pathway of JNK activation. Thisinhibition of JNKK2 is sufficient to account for the effects of Gadd45βon MAPK signaling, and explains the specificity of these effects for theJNK pathway. Together, the data indicate that Gadd45β suppresses JNKactivation, and thereby apoptosis, induced by TNFα and stress stimuli bydirect targeting of JNKK2. Since Gadd45β is able to bind to and inhibitJNKK2 activity in vitro (FIGS. 20 and 21), Gadd45β likely blocks thiskinase directly, either by inducing conformational changes or sterichindrances that impede kinase activity. These findings identifyJNKK2/MKK7 as an important molecular target of Gadd45β in the JNKcascade. Under certain circumstances, Gadd45β may also inhibit JNKK1,albeit more weakly than JNKK2. Because ASK1 is essential for sustainedactivation of JNK and apoptosis by TNF-Rs, it is possible that theinteraction between Gadd45, and this MAPKKK is also relevant to JNKinduction by these receptors.

FIG. 23A-B shows that two distinct polypeptide regions in the kinasedomain of JNKK2 are essential for the interaction with Gadd45β. Byperforming GST pull-down assays with GST- and GST-Gadd45β-coated beads,the regions of JNKK2 that are involved in the interaction with Gadd45βwere determined. pBS plasmids encoding various amino-terminaltruncations of JNKK2 were translated in vitro in the presence of³⁵S-metionine, and binding of these peptides to GST-Gadd45β was assayedas described herein (FIG. 23A, Top), JNKK2(1-401; FL), JNKK2(63-401),JNKK2 (91-401), and JNKK2 (132-401) polypeptides strongly interactedwith Gadd45β, in vitro, indicating that the amino acid region spanningbetween residue 1 and 131 is dispensable for the JNKK2 association withGadd45β. However, shorter JNKK2 truncations—namely JNKK2(157-401),JNKK2(176-401), and JNKK2(231-401)—interacted with Gadd45β more weakly,indicating that the amino acid region between 133 and 156 is criticalfor strong binding to Gadd45β. Further deletions extending beyondresidue 244 completely abrogated the ability of the kinase to associatewith Gadd45β, suggesting that the 231-244 region of JNKK2 alsocontributes to binding to Gadd45β.

To provide further support for these findings, carboxy-terminaldeletions of JNKK2 were generated, by programming retyculo-lysatereactions with pBS-JNKK2 templates that had been linearized withappropriate restriction enzymes (FIG. 23B, bottom). Binding assays withthese truncations were performed as described herein. Digestions ofpBS-JNKK2(FL) with SacII (FL), PpuMI, or NotI did not significantlyaffect the ability of JNKK2 to interact with Gadd45β, indicating thatamino acids 266 to 401 are dispensable for binding to this factor.Conversely, digestions with XcmI or BsgI, generating JNKK2(1-197) andJNKK2(1-186) polypeptides, respectively, partly inhibited binding toGadd45β. Moreover, cleavage with BspEI, BspHI, or PflMI, generatingshorter amino terminal polypeptides, completely abrogated this binding.Together these findings indicate that the polypeptide regions spanningfrom amino acids 139 to 186 and 198 to 265 and are both responsible forstrong association of JNKK2 with Gadd45β. The interaction of JNKK2 withGadd45, was mapped primarily to two polypeptides spanning between JNKK2residue 132 and 156 and between residue 231 and 244. JNKK2 might alsocontact Gadd45β through additional amino acid regions.

The finding that Gadd45β directly contacts two distinct amino acidregions within the catalytic domain of JNKK2 provides mechanisticinsights into the basis for the inhibitory effects of Gadd45, on JNKK2.These regions of JNKK2 shares no homology within MEKK4, suggesting thatGadd45β contacts these kinases through distinct surfaces. Since it isnot known to have enzymatic activity (e.g. phosphatase or proteolyticactivity), and its binding to JNKK2 is sufficient to inhibit kinasefunction, in vitro, Gadd45β might block JNKK2 through directinterference with the catalytic domain, either by causing conformationalchanges or steric hindrances that inhibit kinase activity or access tosubstrates. With regard to this, the 133-156 peptide region includesamino acid K149—a critical residue for kinase activity—thereby providinga possible mechanism for the potent inhibition of JNKK2 by Gadd45β.

FIG. 24A-B shows the Gadd45β amino acid region spanning from residue 69to 104 is essential for interaction with JNKK2 (see also FIGS. 36 and37). To identify the region of Gadd45β that mediated the associationwith JNKK2, GST pull-down experiments were performed. Assays wereperformed using standard protocols and GST-JNKK2- or GST-coated beads.pBS plasmids encoding progressively shorter amino-terminal deletions ofGadd45β were translated in vitro and labeled with ³⁵S_metionine (FIG.24A). Murine Gadd45β(1-160; FL), Gadd45β(41-160), Gadd45β(60-160), andGadd45β(69-160) polypeptides strongly interacted with JNKK2, whereasGadd45β(87-160) bound to the kinase only weakly. In contrast,Gadd45β(114-160) was unable to associate with JNKK2.

To confirm these findings, a series of carboxy-terminal Gadd45βtruncations were generated by programming in vitrotranscription/translation reactions with appropriately linearizedpBS-Gadd45β plasmids (FIG. 24B). Although digestion of pBS-Gadd45β withNgoMI did not affect Gadd45 binding to JNKK2, digestions with SphI andEcoRV, generating Gadd45β(1-95) and Gadd45β(1-68), respectively,progressively impaired Gadd45β affinity for JNKK2. Indeed, the latterpolypeptides were unable to associate with JNKK2. Together the dataindicate that the Gadd45β polypeptide spanning from residue 69 to 104participates in an interaction with JNKK2.

FIG. 25 show the amino acid region spanning between residue 69 and 113is needed for the ability of Gadd45β to suppress TNFα-induced apoptosis(but see FIGS. 36-37). By performing mutational analyses, the domain ofGadd45β that is required for the blocking of TNFα-induced killing wasmapped to the 69-113 amino acid region. Upon expression in RelA^(−/−)cells, GFP-Gadd45β(69-160) and GFP-Gadd45β(1-113) exhibitedanti-apoptotic activity against TNFα that was comparable to that offull-length GFP-Gadd45 P. In contrast, in these assays, GFP proteinsfused to Gadd45β(87-160) or Gadd45β(1-86) had only modest protectiveeffects. Shorter truncations had virtually no effect on cell survival,indicating that the Gadd45β region spanning between amino acids 69 and113 provides cytoprotection, and that the adjacent 60-68 regioncontributes only modestly to this activity.

This amino acid region contains the domain of Gadd that is alsoresponsible for the interaction with JNKK2. This is consistent with thenotion that the protective activity of Gadd45β is linked to its abilityto bind to JNKK2 and suppress JNK activation.

FIG. 26 shows that Gadd45β physically interacts with kinases in the JNKpathway. a, b, Western blots with anti-FLAG immunoprecipitates (top) ortotal lysates (middle and bottom) from 293 cells showing Gadd45βassociation with ASK1, MEKK4, and MKK7. c, Pull-down assays using GST-or GST-Gadd45β-coated beads and ³⁵S-labeled, in vitro translatedproteins. Shown is 40% of the inputs.

FIG. 27 shows that Gadd45β and NF-κB specifically inhibit MKK7, in vivo.a-e, Western blots with antibodies against phosphorylated (P) or totalkinases and kinase assays (K.A.) showing MAPKK and MAPKKK activation byTNFα or P/I in (a-c) IκBαM-Hygro and IκBαM-Gadd45β clones and in (d, e)Neo and IκBαM 3DO clones. a, d, MKK7 phosphorylation (P-MKK7) wasmonitored by combined immunoprecipitation (anti-P-MKK7 antibodies) andWestern blotting (anti-total MKK7 antibodies).

FIG. 28 shows that Gadd45β is a direct inhibitor of MKK7. a,Immunoprecipitations followed by Western blots showing physicalassociation of endogenous Gadd45, and MKK7 (top) in 3DO cells treatedwith P/I (2 hours) or left untreated (US). Protein levels are shown(bottom). b, g, Coomassie brilliant blue staining (CS) showing purity ofthe proteins used in (c) and (d, e), respectively. c, In vitro pull-downassays with purified proteins showing direct interaction betweenHis₆/T7-Gadd45β (His₆disclosed as SEQ ID NO: 46) and GST-MKK7.Precipitated GST proteins and bound His₆/T7-tagged proteins(His₆disclosed as SEQ ID NO: 46) were visualized by CS and Western blotting(WB) with anti-T7 antibodies, respectively. Inputs of His₆/T7-taggedproteins (His₆ disclosed as SEQ ID NO: 46) are indicated. The fractionof His₆/T7-Gadd45β (His₆ disclosed as SEQ ID NO: 46) and His₆/T7-JIP1(His₆ disclosed as SEQ ID NO: 46) binding to GST-MKK7 (expressed asarbitrary units [a.u.]; left) was calculated relatively to a standardcurve generated with known protein concentrations¹⁹. d, e, Kinase assaysshowing specific inhibition of active MKK7 by purified GST-Gadd45β andHis₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46), in vitro. FLAG-taggedkinases were immunoprecipitated from 293 cells treated with TNFα (10minutes) or left untreated and pre-incubated with the indicatedconcentrations of Gadd45 β polypeptides. f, Western blots showingexogenous kinase levels in 293 cells.

FIG. 29 shows that MKK7 contacts Gadd45β through two petidic regions inits catalytic domain. a, c, e, are schematic representations of the MKK7N- and C-terminal truncations and peptides, respectively, used forbinding assays. Interaction regions are shaded in gray. b, d, f, GST arepull-downs showing GST-Gadd45β binding to the indicated 35S-labeled, invitro translated MKK7 products. Shown is 40% of the inputs. g, is anamino acid sequence of Gadd45β-interacting peptides 1 (SEQ ID NO: 4) and7 (SEQ ID NO: 5). K149 is highlighted.

FIG. 30 shows that peptide 1 impairs the ability of Gadd45 β (and NF-κB)to suppress JNK activation and apoptosis induced by TNFα. a, Kinaseassay (K.A.) showing that binding to peptidic region 1 is required forMKK7 inactivation by Gadd45 β. FLAG-MKK7 was immunoprecipitated fromTNFα-treated (10 minutes) 293 cells. b, c, are apoptosis assays showingthat peptide 1 promotes killing by TNFα in IκBαM-Gadd45β and Neo clones,respectively. Values (expressed as arbitrary units) were obtained bysubtracting background values with untreated cells from values withTNFα-treated cells, and represent the mean (+/−standard deviation) ofthree experiments.

FIG. 31 (A-D) shows nucleotide and amino acid sequences of human andmurine JNKK2 (SEQ ID NOS: 49-52, respectively, in order of appearance.).

FIG. 32 shows that Gadd45β blocks MKK7 by contacting a peptidic regionin its catalytic domain. a, Schematic representation of the MKK7peptides used for binding assays. Interaction regions are in gray. b, d,e, GST pull-down assays showing GST-Gadd45β binding to the indicated³⁵S-labeled, in vitro translated MKK7 products. 40% of the inputs isshown (b, d, e, ATP was used as indicated. c, Amino acid sequence ofGadd45β-interacting, peptides 1 (SEQ ID NO: 4) and 7 (SEQ ID NO: 5), andpeptide 1 mutants (SEQ ID NOS: 6-12, respectively, in order ofappearance.) used in (d). K149 is marked by an asterisk. Amino acidsinvolved in binding to Gadd45β are in gray, and darkness correlates withtheir apparent relevance for this binding. f, Kinase assay (K.A.)showing that binding to peptidic region 1 is required for MKK7inactivation by Gadd45β. FLAG-MKK7 was immunoprecipitated fromTNFα-treated (10 minutes) 293 cells. The underlined and bold amino acidsin c represent inserted amino acids that were not present in theoriginal p1 (132-156).

FIG. 33 shows that Gadd45β-mediated suppression of MKK7 is required toblock TNFα-induced apoptosis. A-B, Apoptosis assays showing that peptide1 effectively promotes killing by TNFα in IκBαM-Gadd45β and Neo 3DOclones, respectively. C-D, Apoptosis assays showing that both peptide 1and peptide 2 can facilitate TNFα-induced cytotoxicity in wild-typeMEFs, and that only peptide 2 promotes this killing in Gadd45β nullMEFs, respectively. (C-D), MEFs were from twin embryos and were used atpassage (p)₄. A-D, Values (expressed as arbitrary units) were obtainedby subtracting background values with untreated cells from values withTNFα-treated cells, and represent the mean (+/−standard deviation) ofthree experiments.

FIG. 34 shows that synthetic, FITC-labeled TAT peptides enter cells withcomparable efficiencies. a-d, FCM (a, c) and confocal microscopy (b, d)analyses of 3DO cells after a 20-minute incubation with DMSO (Ctr) orthe indicated peptides (5 □M). a, c, Depicted in the histograms are theoverlaid profiles of DMSO-(gray) and peptide-treated (black) cells. e,Amino acid sequence of the peptide 1 mutants that were fused to TAT forin vivo studies (SEQ ID NOS: 4, 7, 9 and 10, respectively, in order ofappearance.). Note that Ala-II* contains the R140 mutation, not presentin Ala-II, and that in Ala-V*, mutations are shifted of 1 amino acid tothe C-terminus as compared to Ala-V (see FIG. 32 c). Ala-IV* isidentical, in its MKK7-mimicking portion, to Ala-IV.

FIG. 35 shows that peptides that interfere with Gadd45β binding to MKK7blunt the Gadd45β protective activity against TNFα.

FIG. 36 shows that the 69-86 amino acid region of Gadd45, is sufficientto bind to MKK7 in vitro.

FIG. 37 shows that the Gadd45β-mediated inhibition of MKK7 requires apolypeptide region of Gadd45β, including the section between amino acids60 and 86 (SEQ ID NOS: 36-44, respectively, in order of appearance.).

DETAILED DESCRIPTION

The JNK pathway is a focus for control of pathways leading to programmedcell death.

The present invention facilitates development of new methods andcompositions for ameliorating of diseases. Indeed, the observation thatthe suppression of JNK represents a protective mechanism by NF-□Bsuggests that apoptosis of unwanted self-reactive lymphocytes and otherpro-inflammatory cells (e.g. macrophages) at the site ofinflammation—where there are high levels of TNFα—may be augmented byinterfering with the ability of NF-□B to shut down JNK activation.Potential means for achieving this interference include, for instance,using blockers of Gadd45 B and agents that interfere JNKK2-interactingfactors. One such agent is a peptide NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH(SEQ ID NO: 1).

Like Fas, TNF-R1 is also involved in host immune surveillancemechanisms. Thus, in another aspect of the invention, the agents mightprovide a powerful new adjuvant in cancer therapy.

Conversely, an enhancement of cell survival by the down-modulation ofJNK will have beneficial effects in degenerative disorders andimmunodeficiencies, conditions that are generally characterized byexaggerated cell death.

The invention allows design of agents to modulate the JNK pathway e.g.cell permeable, fusion peptides (such as TAT-fusion peptides)encompassing the amino acid regions of JNKK2 that come into directcontact with Gadd45 P. These peptides will effectively compete withendogenous Gadd45 β proteins for binding to JNKK2. In addition, thesefindings allow design of biochemical assays for the screening oflibraries of small molecules and the identification of compounds thatare capable to interfere with the ability of Gadd45β to associate withJNKK2. Both these peptides and these small molecules are able to preventthe ability of Gadd45β, and thereby of NF-κB, to shut down JNKactivation, and ultimately, to block apoptosis. These compounds areuseful in the treatment of human diseases, including chronicinflammatory and autoimmune conditions and certain types of cancer.

The new molecular targets for modulating the anti-apoptotic activity ofNF-κB, are useful in the treatment of certain human diseases. Theapplication of these findings appears to pertain to the treatment of twobroadly-defined classes of human pathologies: a) immunological disorderssuch as autoimmune and chronic inflammatory conditions, as well asimmunodeficiencies; b) certain malignancies, in particular those thatdepend on NF-κB for their survival—such as breast cancer, HL, multiplemyeloma, and DLBCL.

A question was whether JNK played a role in TNF-R-induced apoptosis.Confirming findings in NF-κB-deficient cells, evidence presented hereinnow conclusively demonstrated that JNK activation is obligatory not onlyfor stress-induced apoptosis, but also for efficient killing by TNFα. Itwas shown that fibroblasts lacking ASK1—an essential component of theTNF-R pathway signaling to JNK (and p38)—are resistant to killing byTNFα. Foremost, JNK1 and JNK2 double knockout MEFs exhibit aprofound—albeit not absolute—defect in the apoptotic response tocombined cytotoxic treatment with TNFα and cycloheximide. Moreover, itwas shown that the TNFα homolog of Drosophila, Eiger, completely dependson JNK to induce death, whereas it does not require the caspase-8homolog, DREDD. Thus, the connection to JNK appears to be a vestigialremnant of a primordial apoptotic mechanism engaged by TNFα, which onlylater in evolution begun to exploit the FADD-dependent pathway toactivate caspases.

How can then the early observations with DN-MEKK1 be reconciled withthese more recent findings? Most likely, the key lies in the kinetics ofJNK induction by TNF-Rs. Indeed, apoptosis has been associated withpersistent, but not transient JNK activity. This view is supported bythe recent discovery that JNK activation is apoptogenic on itsown—elegantly demonstrated by the use of MKK7-JNK fusion proteins, whichresult in constitutively active JNK in the absence of extrinsic cellstimulation. Unlike UV and other forms of stress, TNFα causes onlytransient induction of JNK, and in fact, this induction normally occurswithout significant cell death, which explains why JNK inhibition byDN-MEKK1 mutants has no effect on cell survival. JNK pro-apoptoticactivity is instead unmasked when the kinase is allowed to signalchronically, for instance by the inhibition of NF-κB.

The exact mechanism by which JNK promotes apoptosis is not known. Whilein some circumstances JNK-mediated killing involves modulation of geneexpression, during challenge with stress or TNFα, the targets of JNKpro-apoptotic signaling appear to be already present in the cell.Killing by MKK7-JNK proteins was shown to require Bax-like factors ofthe Bcl-2 group; however, it is not clear that these factors are directtargets of JNK, or that they mediate JNK cytotoxicity during TNF-Rsignaling.

I. Activation of the JNK Cascade is Required for Efficient Killing byDRs (TNF-R1, Fas, and TRAIL-Rs), and the Suppression of this Cascade isCrucial to the Protective Activity of NF-κB A. TNF-Rs-Induced Apoptosis

The JNK and NF-κB pathways—almost invariably co-activated by cytokinesand stress—are intimately linked. The blocking of NF-κB activation byeither the ablation of the NF-κB subunit RelA or expression of the IκBαMsuper-inhibitor hampers the normal shut down of JNK induction by TNF-R(FIGS. 5 a and 5 b). Indeed, the down-regulation of the JNK cascade byNF-κB is needed for suppression of TNFα-induced apoptosis, as shown bythe finding that inhibition of JNK signaling by various means rescuesNF-κB-deficient cells from TNFα-induced apoptosis (FIGS. 5 d and 5 e).In cells lacking NF-κB, JNK activation remains sustained even afterprotective treatment with caspase inhibitors, indicating that theeffects of NF-κB on the JNK pathway are not a secondary consequence ofcaspase inhibition. Thus, NF-κB complexes are true blockers of JNKactivation. These findings define a novel protective mechanism by NF-κBand establish a critical role for JNK (and not for p38 or ERK) in theapoptotic response to TNFα (see FIG. 18).

B. Fas-Induced Apoptosis

Although ASK1^(−/−) and JNK null fibroblasts are protected against thecytotoxic effects of TNFα, these cells retain normal sensitivity toFas-induced apoptosis, which highlights a fundamental difference betweenthe apoptotic mechanisms triggered by Fas and TNF-R. Nevertheless, incertain cells (e.g. B cell lymphomas), JNK is also involved in theapoptotic response to Fas triggering. Indeed, the suppression of JNK byvarious means, including the specific pharmacological blocker SP600125,rescues BJAB cells from Fas-induced cytotoxicity (FIG. 14). Consistentwith this observation, in these cells, killing by Fas is also almostcompletely blocked by over-expression of Gadd45β (FIG. 13B). Together,these findings indicate that JNK is required for Fas-induced apoptosisin some circumstance, for instance in type 2 cells (e.g. BJAB cells),which require mitochondrial amplification of the apoptotic signal toactivate caspases and undergo death.

Like TNF-Rs, Fas plays an important role in the host immune surveillanceagainst cancerous cells. Of interest, due to the presence ofconstitutively high NF-κB activity, certain tumor cells are able toevade these immune surveillance mechanisms. Thus, an augmentation of JNKsignaling—achieved by blocking the JNK inhibitory activity of Gadd45β,or more broadly of NF-κB—aids the immune system to dispose of tumorcells efficiently.

Fas is also critical for lymphocyte homeostasis. Indeed, mutations inthis receptor or its ligand, FasL, prevent elimination of self-reactivelymphocytes, leading to the onset of autoimmune disease. Thus, for thetreatment of certain autoimmune disorders, the inhibitory activity ofGadd45β on JNK may serve as a suitable target.

C. TRAIL-R-Induced Apoptosis

Gadd45β also blocks TRAIL-R-involved in apoptosis (FIG. 1A), suggestingthat JNK plays an important role in the apoptotic response to thetriggering of this DR. The finding that JNK is required for apoptosis byDRs may be exploited for cancer therapy. For example, the sensitivity ofcancer cells to TRAIL-induced killing by adjuvant treatment is enhancedwith agents that up-regulate JNK activation. This can be achieved byinterfering with the ability of Gadd45β or NF-κB to block TRAIL-inducedJNK activation. This finding may also provide a mechanism for thesynergistic effects of combined anti-cancer treatment because JNKactivation by genotoxic chemotherapeutic drugs may lower the thresholdfor DR-induced killing.

II. The Suppression of JNK Represents a Mechanism by which NF-κBPromotes Oncogenesis and Cancer Chemoresistance

In addition to antagonizing DR-induced killing, the protective activityof NF-κB is crucial to oncogenesis and chemo- and radio-resistance incancer. However, the bases for this protective activity is poorlyunderstood. It is possible that the targeting of the JNK cascaderepresents a general anti-apoptotic mechanism by NF-κB, and indeed,there is evidence that the relevance of this targeting by NF-κB extendsto both tumorigenesis and resistance of tumor cells to anti-cancertherapy. During malignant transformation, cancer cells must adoptmechanisms to suppress JNK-mediated apoptosis induced by oncogenes, andat least in some cases, this suppression of apoptotic JNK signalingmight involve NF-κB. Indeed, while NF-κB activation is required to blocktransformation-associated apoptosis, non-redundant components of the JNKcascade such as MKK4 and BRCA1 have been identified as tumorsuppressors.

Well-characterized model systems of NF-κB-dependent tumorigenesis,including such as breast cancer cells provide insight into mechanism ofaction. Breast cancer cell lines such as MDA-MD-231 and BT-20, which areknown to depend on NF-κB for their survival, can be rescued fromapoptosis induced by NF-κB inhibition by protective treatment with theJNK blocker SP600125 (FIG. 17). Thus, in these tumor cells, the ablationof JNK can overcome the requirement for NF-κB, suggesting thatcytotoxicity by NF-κB inactivation is associated with anhyper-activation of the JNK pathway, and indicates a role for thispathway in tumor suppression. Gadd45β mediates the protective effects ofNF-κB during oncogenesis and cancer chemoresistance, and is a noveltarget for anti-cancer therapy.

With regard to chemoresistance in cancer, apoptosis by genotoxicstress—a desirable effect of certain anti-cancer drugs (e.g.daunorubicin, etopopside, and cisplatinum)—requires JNK activation,whereas it is antagonized by NF-κB. Thus, the inhibition of JNK is amechanism by which NF-κB promotes tumor chemoresistance. Indeed,blockers of NF-κB are routinely used to treat cancer patients such aspatients with HL and have been used successfully to treat otherwiserecalcitrant malignancies such as multiple myeloma. However, theseblockers (e.g. glucocorticoids and proteosome inhibitors) can onlyachieve a partial inhibition of NF-κB, and when used chronically,exhibit considerable side effects, including immune suppressive effects,which limit their use in humans. Hence, as discussed with DRs, in thetreatment of certain malignancies, it is beneficial to employ, ratherthan NF-κB-targeting agents, therapeutic agents aimed at blocking theanti-apoptotic activity of NF-κB. For instance, a highly effectiveapproach in cancer therapy may be the use of pharmacological compoundsthat specifically interfere with the ability of NF-κB to suppress JNKactivation. These compounds not only enhance JNK-mediated killing oftumor cells, but allow uncoupling of the anti-apoptotic andpro-inflammatory functions of the transcription factor. Thus, unlikeglobal blockers of NF-κB, such compounds lack immunosuppressive effects,and thereby represent a promising new tool in cancer therapy. A suitabletherapeutic target is Gadd45β itself, because this factor is capable ofinhibiting apoptosis by chemotherapeutic drugs (FIGS. 3D and 3E), andits induction by these drugs depends on NF-κB (FIG. 2D). With regard tothis, the identification of the precise mechanisms by which Gadd45β andNF-κB block the JNK cascade (i.e. the testing of JNKK2) opens up newavenues for therapeutic intervention in certain types of cancer, inparticular in those that depend on NF-κB, including tumors driven byoncogenic Ras, Bcr-Abl, or EBV-encoded oncogenes, as well as late stagetumors such as HL, DLBCL, multiple myeloma, and breast cancers.

III. Gadd45β Mediates the Inhibition of the JNK Cascade by NF-κB A.Gadd45β Mediates the Protective Effects of NF-κB against DR-InducedApoptosis

Cytoprotection by NF-κB involves activation of a program of geneexpression. Pro-survival genes that mediate this important function ofNF-κB were isolated. In addition to gaining a better understanding ofthe molecular basis for cancer, the identification of these genesprovides new targets for cancer therapy. Using a functional screen inNF-κB/RelA null cells, Gadd45β was identified as a pivotal mediator ofthe protective activity of NF-κB against TNFα-induced killing. gadd45βis upregulated rapidly by the cytokines through a mechanism thatrequires NF-κB (FIGS. 2A and 2B), antagonizes TNFα-induced killing (FIG.1F), and blocks apoptosis in NF-κB null cells (FIGS. 1A, 1C, 1D, 3A and3B). Cytoprotection by Gadd45β involves the inhibition of the JNKpathway (FIGS. 4A, 4C and 4D), and this inhibition is central to thecontrol of apoptosis by NF-κB (FIGS. 5A, 5B, 5D and 5E). Expression ofGadd45β in cells lacking NF-κB completely abrogates the JNK activationresponse to TNFα, and inhibition of endogenous proteins by anti-sensegadd45β hinders the termination of this response (FIG. 4D). Gadd45β alsosuppresses the caspase-independent phase of JNK induction by TNFα, andhence, is a bona fide inhibitor of the JNK cascade (FIGS. 4A and 4C).There may be additional NF-κB-inducible blockers of JNK signaling.

Activation of gadd45β by NF-κB was shown to be a function of threeconserved κB elements located at positions −447/−438 (κB-1), −426/−417(κB-2), and −377/−368 (κB-3) of the gadd45β promoter (FIGS. 8, 9A, 9B,10A, 10B, and 11). Each of these sites binds to NF-κB complexes in vitroand is required for optimal promoter transactivation (FIGS. 12A, 12B,and 12C). Together, the data establish that Gadd45β is a novelanti-apoptotic factor, a physiologic inhibitor of JNK activation, and adirect transcriptional target of NF-κB. Hence, Gadd45β mediates thetargeting of the JNK cascade and cytoprotection by NF-κB.

The protective activity of Gadd45β extends to DRs other than TNF-Rs,including Fas and TRAIL-Rs. Expression of Gadd45β dramatically protectedBJAB cells from apoptosis induced by the triggering of either one ofthese DRs, whereas death was effectively induced in control cells (FIGS.13B and 13A, respectively). Remarkably, in the case of Fas, protectionby Gadd45β was nearly complete. Similar to TNF-R1, the protectiveactivity of Gadd45β against killing by Fas, and perhaps by TRAIL-Rs,appears to involve the inhibition of the JNK cascade (FIGS. 13A, 13B and14). Thus, Gadd45β is a new target for modulating DR-induced apoptosisin various human disorders.

B. Gadd45β is a Potential Effector of the Protective Activity of NF-κBduring Oncogenesis and Cancer Chemoresistance

The protective genes that are activated by NF-κB during oncogenesis andcancer chemoresistance are not known. Because it mediates JNK inhibitionand cytoprotection by NF-κB, Gadd45β is a candidate. Indeed, as with thecontrol of DR-induced apoptosis, the induction of gadd45β represents ameans by which NF-κB promotes cancer cell survival. In 3DO tumor cells,Gadd45β expression antagonized killing by cisplatinum and daunorubicin(FIGS. 3D and 3E)—two genotoxic drugs that are widely-used inanti-cancer therapy. Thus, Gadd45, blocks both the DR and intrinsicpathways of caspase activation found in mammalian cells. Since apoptosisby genotoxic agents requires JNK, this latter protective activity ofGadd45β might also be explained by the inhibition of the JNK cascade. In3DO cells, gadd45β expression was strongly induced by treatment witheither daunorubicin or cisplatinum, and this induction was almostcompletely abolished by the IκBαM super-repressor (FIG. 2D), indicatingthat gadd45β activation by these drugs depends on NF-κB. Hence, Gadd45βmay block the efficacy of anti-tumor therapy, suggesting that itcontributes to NF-κB-dependent chemoresistance in cancer patients, andthat it represents a new therapeutic target.

Given the role of JNK in tumor suppression and the ability of Gadd45β toblock JNK activation, Gadd45β also is a candidate to mediate NF-κBfunctions in tumorigenesis. Indeed, expression patterns suggest thatGadd45β may contribute to NF-κB-dependent survival in certain late stagetumors, including ER breast cancer and HL cells. In cancer cells, butnot in control cells such as less invasive, ER⁺ breast cancers, gadd45βis expressed at constitutively high levels (FIG. 16), and these levelscorrelate with NF-κB activity.

C. Identification of the Mechanisms by which Gadd45β Blocks JNKActivation: the Targeting of JNKK2/MKK7

Neither Gadd45β nor NF-κB affect the ERK or p38 cascades (FIG. 4C),suggesting that these factors block JNK signaling downstream of theMAPKKK module. Consistent with this notion, the MAPKK, JNKK2/MKK7—aspecific activator of JNK and an essential component of the TNF-Rpathway of JNK activation were identified as the molecular target ofGadd45β in the JNK cascade.

Gadd45β was previously shown to associate with MEKK4. However, sincethis MAPKKK is not activated by DRs, the functional consequences of thisinteraction were not further examined. Thus, to begin to investigate themechanisms by which Gadd45β controls JNK induction by TNF-R, Gadd45β wasexamined for the ability to physically interact with additional kinases,focusing on those MAPKKKs, MAPKKs, and MAPKs that have been reported tobe induced by TNF-Rs. Co-immunoprecipitation assays confirmed theability of Gadd45β to bind to MEKK4 (FIG. 19). These assays also showedthat Gadd45β is able to associate with ASK1, but not with otherTRAF2-interacting MAPKKKs such as MEKK1, GCK, and GCKR, or additionalMAPKKK that were tested (e.g. MEKK3) (FIG. 19). Notably, Gadd45β alsointeracted with JNKK2/MKK7, but not with the other JNK kinase,JNKK1/MKK4, or with any of the other MAPKKs and MAPKs under examination,including the two p38-specific activators MKK3b and MKK6, and the ERKkinase MEK1 (FIG. 19). In vitro GST pull-down experiments have confirmeda strong and direct interaction between Gadd45β and JNKK2, as well as amuch weaker interaction with ASK1 (FIG. 20). They also uncovered a veryweak association between Gadd45β and JNKK1 (FIG. 20).

Gadd45β is a potent inhibitor of JNKK2 activity. This has been shownboth in in vitro assays (FIG. 22A), using recombinant Gadd45, proteins,and in in vivo assays, using lysates of 3DO clones (FIG. 22A). Theeffects of Gadd45β on JNKK2 activity are specific, because even whenused at high concentrations, this factor is unable to inhibit eitherJNKK1, MKK3b, or—despite its ability to bind to it—ASK1 (FIGS. 21B, 21C,22A and 22B). This inhibition of JNKK2 is sufficient to account for theeffects of Gadd45β on MAPK signaling, and likely explains thespecificity of these effects for the JNK pathway. Together, the dataindicate that Gadd45β suppresses JNK activation, and thereby apoptosis,induced by TNFα and stress stimuli by directly targeting JNKK2 (FIGS.21A and 22A). Consistent with the notion that it mediates the effects ofNF-κB on the JNK cascade, Gadd45β and NF-κB have similar effects on MAPKactivation by TNFα, in vivo (FIG. 4C). Because ASK1 is essential forsustained activation of JNK and apoptosis by TNF-Rs, it is possible thatthe interaction between Gadd45β and this MAPKKK is also relevant to JNKinduction by these receptors.

By performing GST pull-down experiments using either GST-Gadd45β orGST-JNKK2 and several N- and C-terminal deletion mutants of JNKK2 andGadd45β, respectively, the kinase-binding surfaces(s) of Gadd45β (FIGS.24A and 24B) and the Gadd45β-binding domains of JNKK2 (FIGS. 23A and23B) were identified (see also FIGS. 36 and 37). Gadd45β directlycontacts two distinct amino acid regions within the catalytic domain ofJNKK2 (FIGS. 23A and 23B), which provides important mechanistic insightsinto the basis for the inhibitory effects of Gadd45β on JNKK2. Theseregions of JNKK2 share no homology within MEKK4, suggesting that Gadd45βcontacts these kinases through distinct surfaces. Since it is not knownto have enzymatic activity (e.g. phosphatase or proteolytic activity),and its binding to JNKK2 is sufficient to inhibit kinase function, invitro (FIG. 21A), Gadd45β might block JNKK2 through direct interferencewith the catalytic domain, either by causing conformational changes orsteric hindrances that inhibit kinase activity or access to substrates.

By performing mutational analyses, a domain of Gadd45β that isresponsible for the blocking of TNFα-induced killing was mapped (FIG.25). Cytoprotection assays in RelA^(−/−) cells have shown thatGFP-Gadd45β(69-160) and GFP-Gadd45β(1-113) exhibit anti-apoptoticactivity against TNFα that is comparable to that of full-lengthGFP-Gadd45β while GFP proteins fused to Gadd45β(87-160) or Gadd45β(1-86)have only modest protective effects. Shorter truncations have virtuallyno effect on cell survival (FIG. 25), indicating that the Gadd45β regionspanning between amino acids 69 and 113 facilitating cytoprotection.

This same amino acid region containing Gadd45β domain (69-104) that isessential for the Gadd45β interaction with JNKK2 (FIGS. 24A and 24B).This is consistent with the notion that the protective activity ofGadd45β is linked to its ability to bind to JNKK2 and suppress JNKactivation. Of interest, these findings now allow the design of cellpermeable, TAT-fusion peptides encompassing the amino acid regions ofJNKK2 that come into direct contact, with Gadd45β. It is expected thatthese peptides can effectively compete with endogenous Gadd45β proteinsfor binding to JNKK2. In addition, these findings allow to designbiochemical assays for screening libraries of small molecules andidentifying compounds that are capable of interfering with the abilityof Gadd45β to associate with JNKK2. Both these peptides and these smallmolecules prevent the ability of Gadd45β, and thereby of NF-κB, to shutdown JNK activation, and ultimately, to block apoptosis. As discussedthroughout this summary, these compounds might find useful applicationin the treatment of human diseases, including chronic inflammatory andautoimmune conditions and certain types of cancer.

EXAMPLES

The following examples are included to demonstrate embodiments of theinvention. It should be appreciated by those of skill in the art thattechniques disclosed in the examples which follow represent techniquesdiscovered by the inventor to function well in the practice of theinvention. However, those of skill in the art should, in light of thepresent disclosure, appreciate that many changes can be made in thespecific embodiments which are disclosed and still obtain a like orsimilar result without departing from the spirit and scope of theinvention.

Example 1 Identification of Gadd45β as Novel Antagonist of TNFR-InducedApoptosis

Functional complementation of RelA−/− fibroblasts which rapidly undergoapoptosis when treated with TNFα (Beg and Baltimore, 1996), was achievedby transfection of cDNA expression libraries derived fromTNFα-activated, wild-type fibroblasts. A total of four consecutivecycles of library transfection, cytotoxic treatment with TNFα, andepisomal DNA extraction were completed, starting from more than 4×10⁶independent plasmids.

After selection, ˜200 random clones were analyzed in transienttransfection assays, with 71 (35%) found to significantly protectRelA-null cells from TNFα-induced death. Among these were cDNAs encodingmurine RelA, cFLIP, and dominant negative (DN) forms of FADD, which hadbeen enriched during the selection process, with RelA representing 3.6%of the newly-isolated library. Thus, the library abounded in knownregulators of TNFR-triggered apoptosis (Budihardjo et al., 1999).

One of the cDNAs that scored positive in cytoprotection assays encodedfull-length Gadd45β, a factor that had not been previously implicated incellular responses to TNFα Gadd45β inserts had been enriched 82 foldsafter two cycles of selection, reaching an absolute frequency of 0.41%.The above experiment shows that Gadd45β is a novel putativeanti-apoptotic factor.

To confirm the above findings, pEGFP-Gadd45β, pEGFP-RelA, or insert-lesspEGFP constructs were tested in transient transfection assays in RelA−/−fibroblasts. Whereas cells expressing control GFP proteins were, asexpected, highly susceptible to TNFα-induced death, whereas in contrast,cells that had received pEGFP-Gadd45β were dramatically protected formapoptosis-exhibiting a survival rate of almost 60% after an 8-hourtreatment versus 13% in control cultures (FIG. 1A). As shown previously,with pEGFP-RelA the cell rescue was virtually complete (Beg andBaltimore, 1996).

To determine whether the activity of Gadd45β was cell type-specific anadditional cellular model of NK-κB deficiency was generated, where 3DO Tcell hybridomas were forced to stably express IκBαM, a variant of theIκBα inhibitor that effectively blocks the nuclear translocation ofNF-κB (Van Antwerp et al., 1996).

In the presence of the repressor, 3DO cells became highly sensitive toTNFα-induced killing, as shown by nuclear propidium iodide (PI)staining, with the degree of the toxicity correlating with IκBαM proteinlevels (FIG. 1B, lower panels). Neo control cells retained instead, fullresistance to the cytokine. Next, constructs expressing full-lengthGadd45β, or empty control vectors (Hygro) were stably introduced intothe 3DO—IκBαM-25 line, which exhibited the highest levels of IκBαM (FIG.1B). Although each of 11 IκBαM-Hygro clones tested remained highlysusceptible to TNFα, clones expressing Gadd45β became resistant toapoptosis, with the rates of survival of 31 independent IκBαM-Gadd45βclones correlating with Gadd45β protein levels (FIGS. 1C and 1D,representative lines expressing high and low levels of Gadd45β andIκBαM-Hygro controls). The protective effects of Gadd45β were mostdramatic at early time points, when viability of some IκBαM-Gadd45βlines was comparable to that of Neo clones (FIGS. 1C and 1D, 8 hours).In the IκBαM-Gadd45β-33 line, expressing high amounts of Gadd45β, thefrequency of cell death was only 15% higher than in Neo controls even at24 hours (FIG. 1C). Thus, Gadd45β is sufficient to temporarilycompensate for the lack of NF-κB.

Further, IκBαM-Gadd45β cells retained protein levels of IκBαM that weresimilar or higher than those detected in sensitive IκBαM clones (FIG.1D, lower panels) and that were sufficient to completely block NF-κBactivation by TNFα, as judged by electrophoretic mobility shift assays(EMSAs; FIG. 1E). Hence, as also seen in RelA−/− cells, Gadd45β blocksapoptotic pathways by acting downstream of NF-κB complexes.

Example 2 Gadd45 is a Physiologic Target of NF-κB

Gadd45β can be induced by cytokines such as IL-6, IL-18, and TGFβ, aswell as by genotoxic stress (Zhang et al., 1999; Yang et al., 2001; Wanget al., 1999b). Because the NF-κB anti-apoptotic function involves geneactivation, whether Gadd45β was also modulated by TNFα was determined.As shown in FIG. 2A, cytokine treatment determined a strong and rapidupregulation of Gadd45β transcripts in wild-type mouse embryofibroblasts (MEF). In contrast, in cells lacking RelA, gene inductionwas severely impaired, particularly at early time points (FIG. 2A,compare +/+ and −/− lanes at 0.5 hours). In these cells, induction wasalso delayed and mirrored the pattern of expression of IkβαM a knowntarget of NH-κB (Ghosh et al., 1998), suggesting that the modestinduction was likely due to NF-κB family members other than RelA (i.e.,Rel). Gadd45α was not activated by TNFα, while Gadd45γ was modestlyupregulated in both cell types.

Analogously, Gadd45β was induced by TNFα in parental and Neo 3DO cells,but not in the IκBαM lines (FIG. 2B), with modest activation seen onlyin IκBαM-6 cells, which expressed low levels of the repressor (see FIG.1B). In Neo clones, Gadd45β was also induced by daunorubicin or PMA plusionomycin (P/I; FIGS. 2D and 2C, respectively), treatments that areknown to activate NF-κB (Wang et al., 1996). Again, gene induction wasvirtually abrogated by IκBαM. Gadd45α was unaffected by TNFα treatment,but was upregulated by daunorubicin or P/I, albeit independently ofNF-κB (FIG. 2B, C, D); whereas Gadd45γ was marginally induced by thecytokine only in some lines (FIG. 2B). nfκb1 was used as a positivecontrol of NF-κB-dependent gene expression (Ghosh et al., 1998).

The results establish that gadd45β is a novel TNFα-inducible gene and aphysiologic target of NF-κB. The inspection of the gadd45β promoterrevealed the presence of 3 κB binding sites. EMSAs and mutationalanalyses confirmed that each of these sites was required for optimaltranscriptional activation indicating that gadd45β is also a directtarget of NF-κB. These finding are consistent with a role of gadd45β asa physiologic modulator of the cellular response to TNFα.

Example 3 Endogenous Gadd45β is Required for Survival of TNFα

Gadd45β is a downstream target of NF-κB and exogenous Gadd45β canpartially substitute for the transcription factor during the response toTNFα However, it could be argued that since experiments were carried outin overexpression, cytoprotection might not represent a physiologicfunction of Gadd45β. To address this issue, 3DO clones stably expressingGadd45β in anti-sense orientation were generated. The inhibition ofconstitutive Gadd45β expression in these clone led to a slightredistribution in the cell cycle, reducing the fraction of cellsresiding in G₂, which might underline previously proposed roles ofGadd45 proteins in G₂/M checkpoints (Wang et al., 1999c). Despite theirability to activate NF-κB, cells expressing high levels of anti-senseGadd45β (AS-Gadd45β) exhibited a marked susceptibility to the killing byTNFα plus sub-optimal concentrations of CHX (FIG. 1F). In contrast,control lines carrying empty vectors (AS-Hygro) remained resistant tothe treatment (FIG. 1F). As with the alterations of the cell cycle,cytotoxicity correlated with high levels of anti-sense mRNA. The dataindicate that, under normal circumstances, endogenous Gadd45β isrequired to antagonize TNFR-induced apoptosis, and suggest that thesensitivity of NF-κB-null cells to cytokine killing is due, at least inpart, to the inability of these cells to activate its expression.

Example 4 Gadd45β Effectively Blocks Apoptotic Pathways in NF-κB-NullCells

A question was whether expression of Gadd45β affected caspaseactivation. In NF-κ-deficient cells, caspase-8 activity was detected asearly as 4 hours after TNFα treatment, as assessed by the ability of 3DOextracts to proteolyze caspase-8-specific substrates in vitro (FIG. 3A,IκBαM and IκBαM-Hygro). This coincided with the marked activation ofdownstream caspases such as caspase-9, -2, -6, and -3/7. As previouslyreported, this cascade of events, including the activation ofprocaspase-8, was completely blocked by NF-κB (Neo; Wang et al., 1998).The cytokine-induced activation of both initiator and executionercaspases was also suppressed in IκBαM-Gadd45β-10 cells expressing highlevels of Gadd45β (FIG. 3A). Although very low caspase-3/7 activity wasdetected in these latter cells by 6 hours (bottom, middle panel), thesignificance of this finding is not clear since there was no sign of theprocessing of either caspase-3 or -7 in Western blots (FIG. 3B). Indeed,in IκBαM-Gadd45β and Neo cells, the cleavage of other procaspases, aswell as of Bid, was also completely inhibited, despite the presence ofnormal levels of protein proforms in these cells (FIG. 3B). Proteolysiswas specific because other proteins, including β-actin, were notdegraded in the cell extracts. Thus, Gadd45, abrogates TNFα-inducedpathways of caspase activation in NF-κB-null cells.

To further define the Gadd45β-dependent blockade of apoptotic pathways,mitochondrial functions were analyzed. In IκBαM and IκBαM-Hygro clones,TNFα induced a drop of the mitochondrial Δψm, as measured by the use ofthe fluorescent dye JC-1. JC-1⁺ cells began to appear in significantnumbers 4 hours after cytokine treatment, reaching 80% by 6 hours (FIG.3C). Thus in NF-κB-null 3DO cells, the triggering of mitochondrialevents and the activation of initiator and executioner caspases occurwith similar kinetics. The ability of Bcl-2 to protect IκBαM cellsagainst TNFα-induced killing indicates that, in these cells, caspaseactivation depends on mitochondrial amplification mechanisms (Budihardjoet al, 1999). In IκBαM-Gadd45β-10 as well as in Neo cells, mitochondrialdepolarization was completely blocked (FIG. 3A). Inhibition was nearlycomplete also in IκBαM-Gadd45β-5 cells, where low caspase activity wasobserved (FIG. 3A). These findings track the protective activity ofGadd45β to mitochondria, suggesting that the blockade of caspaseactivation primarily depends on the ability of Gadd45β to completelysuppress mitochondrial amplification mechanisms. As shown in FIGS. 3Dand 3E, Gadd45β was able to protect cells against cisplatinum anddaunorubicin, suggesting that it might block apoptotic pathways inmitochondria. Consistent with this possibility, expression of thisfactor also protected cells against apoptosis by the genotoxic agentscisplatinum and daunorubicin (FIGS. 3D and 3E, respectively). BecauseGadd45β does not appear to localize to mitochondria, it most likelysuppresses mitochondrial events indirectly, by inhibiting pathways thattarget the organelle.

Example 5 Gadd45β is a Specific Inhibitor of JNK Activation

A question explored was whether Gadd45β affected MAPK pathways, whichplay an important role in the control of cell death (Chang and Karin,2001). In IκBαM-Hygro clones, TNFα induced a strong and rapid activationof JNK, as shown by Western blots with anti-phospho-JNK antibodies andJNK kinase assays (FIGS. 4A and 5A, left panels). Activation peaked at 5minutes, to then fade, stabilizing at sustained levels by 40 minutes.The specific signals rose again at 160 minutes due to caspase activation(FIGS. 4A and 5A). Unlike the early induction, this effect could beprevented by treating cells with the caspase inhibitor zVAD-fik. InIκBαM-Gadd45β cells, JNK activation by TNFα was dramatically impaired ateach time point, despite the presence of normal levels of JNK proteinsin these cells (FIG. 4A, right panels). Gadd45β also suppressed theactivation of JNK by stimuli other than TNFα, including sorbitol andhydrogen peroxide (FIG. 4B). The blockade, nevertheless, was specific,because the presence of Gadd45β did not affect either ERK or p38activation (FIG. 4C). The anti-sense inhibition of endogenous Gadd45βled to a prolonged activation of JNK following TNFR triggering (FIG. 4D,AS-Gadd45, and Hygro), indicating that this factor, as well as otherfactors (see down-regulation in AS-Gadd45β cells) is required for theefficient termination of this pathway. The slightly augmented inductionseen at 10 minutes in AS-Gadd45β cells showed that constitutivelyexpressed Gadd45β may also contribute to the inhibition of JNK (see FIG.2, basal levels of Gadd45β). Gadd45β is a novel physiological inhibitorof JNK activation. Given the ability of JNK to trigger apoptoticpathways in mitochondria, these observations may offer a mechanism forthe protective activity of Gadd45β.

Example 6 Inhibition of the JNK Pathway as a Novel Protective Mechanismby NF-κB

Down-regulation of JNK represents a physiologic function of NF-κB.Whereas in Neo cells, JNK activation returned to near-basal levels 40minutes after cytokine treatment, in IκBαM as well as in IκBαM-Hygrocells, despite down-modulation, JNK signaling remained sustainedthroughout the time course (FIG. 7A; see also FIG. 5A). Qualitativelysimilar results were obtained with RelA-deficient MEF where, unlike whatis seen in wild-type fibroblasts, TNFα-induced JNK persisted atdetectable levels even at the latest time points (FIG. 5B). Thus, aswith Gadd45β, NF-κB complexes are required for the efficient terminationof the JNK pathway following TNFR triggering thus establishing a linkbetween the NF-κB and JNK pathways.

CHX treatment also impaired the down-regulation to TNFα-induced JNK(FIG. 5C), indicating that, in 3DO cells, this process requiresnewly-induced and/or rapidly turned-over factors. Although in somesystems, CHX has been reported to induce a modest activation of JNK (Liuet al., 1996), in 3DO cells as well as in other cells, this agent alonehad no effect on this pathway (FIG. 5C; Guo et al., 1998). The findingsindicate that the NF-κB-dependent inhibition of JNK is most likely atranscriptional event. This function indicates the involvement of theactivation of Gadd45β, because this factor depends on the NF-κB for itsexpression (FIG. 2) and plays an essential role in the down-regulationof TNFR-induced JNK (FIG. 4D).

With two distinct NF-κB-null systems, CXH-treated cells, as well asAS-Gadd45β cells, persistent JNK activation correlated withcytotoxicity, whereas with IκBαM-Gadd45β cells, JNK suppressioncorrelated with cytoprotection. To directly assess whether MAPK cascadesplay a role in the TNFα-induced apoptotic response of NF-κB-null cells,plasmids expressing catalytically inactive mutants of JNKK1 (MKK4; SEK1)or JNKK2 (MKK7), each of which blocks JNK activation (Lin et al., 1995),or of MKK3b, which blocks p38 (Huang et al., 1997), or empty vectorswere transiently transfected along with pEGFP into RelA−/− cells.Remarkably, whereas the inhibition of p38 had no impact on cellsurvival, the suppression of JNK by DN-JNKK2 dramatically rescuedRelA-null cells from TNFα-induced killing (FIG. 5D). JNKK1 is notprimarily activated by proinflammatory cytokines (Davis, 2000), whichmay explain why JNKK1 mutants had no effect in this system. Similarfindings were obtained in 3DO—IκBαM cells, where MAPK pathways wereinhibited by well-characterized pharmacological agents. Whereas, PD98059and low concentrations of SB202190 (5 μM and lower), which specificallyinhibit ERK and p38, respectively, could not antagonize TNFαcytotoxicity, high concentrations of SB202190 (50 μM), which blocks bothp38 and JNK (Jacinto et al., 1998), dramatically enhanced cell survival(FIG. 5E). The data indicate that JNK, but not p38 (or ERK), transducescritical apoptotic signals triggered by TNFR and that NF-κB complexesprotect cells, at least in part, by prompting the down-regulation of JNKpathways.

Example 7 gadd45β is Induced by the Ectopic Expression of RelA, but notRel or p50

The activation of gadd45β by cytokines or stress requires NF-κB, as isdisclosed herein because induction in abolished either by RelA-nullmutations or by the expression of IκBαM, a variant of the IκBα inhibitorthat blocks that nuclear translocation of NF-κB (Van Antwerp et al.,1996). To determine whether NF-κB is also sufficient to upregulategadd45β and, if so, to define which NF-κB family members are mostrelevant to gene regulation, HeLa-derived HtTA-RelA, HtTA-CCR43, andHtTA-p50 cell lines, which express RelA, Rel, and p50, respectively,were used under control of a teracyclin-regulated promoter (FIG. 6).These cell systems were employed because they allow NF-κB complexes tolocalize to the nucleus independently of extracellular signals, whichcan concomitantly activate transcription factors of the NF-κB.

As shown in FIG. 6, the withdrawal of tetracycline prompted a strongincrease of gadd45β mRNA levels in HtTA-RelA cells, with kinetics ofinduction mirroring those of relA, as well as iκbα and p105, two knowntargets of NF-κB. As previously reported, RelA expression inducedtoxicity in these cells (gadph mRNA levels) lead to underestimation ofthe extent of gadd45β induction. Conversely, gadd45β was only marginallyinduced in HtTA-CCR43 cells, which conditionally express high levels ofRel. iκbα and p105 were instead significantly activated in these cells,albeit to a lesser extent than in the HtTA-RelA line, indicating thattetracycline withdrawal yielded functional Rel-containing complexes. Theinduction of p50, and NF-κB subunit that lacks a defined activationdomain, did not affect endogenous levels of either gadd45β, iκbα, orp105. The withdrawal of tetracycline did not affect gadd45β (or relA,rel, or p105) levels in HtTA control cells, indicating the gadd45βinduction in HtTA-RelA cells was due to the activation of NF-κBcomplexes.

Kinetics of induction of NF-κB subunits were confirmed by Western blotanalyses. Hence gadd45β expression is dramatically and specificallyupregulated upon ectopic expression of the transcriptionally activeNF-κB subunit RelA, but not of p50 or Rel (FIG. 6). These findings areconsistent with the observations with RelA-null fibroblasts describedabove and underscore the importance of RelA in the activation ofgadd45β.

Example 8 gadd45β Expression Correlates with NF-κB Activity in B CellLines

NF-κB plays a critical role in B lynphopoiesis and is required forsurvival of mature B cells. Thus, constitutive and inducible expressionof gadd45β were examined in B cell model systems that had beenwell-characterized from the stand point of NF-κB. Indeed, gadd45β mRNAlevels correlated with nuclear NF-κB activity in these cells (FIG. 7).Whereas gadd45β transcripts could be readily seen in unstimulatedWEHI-231 B cells, which exhibit constitutively nuclear NF-κB, mRNAlevels were below detection in 70Z/3 pre-B cells, which contain insteadthe classical inducible form of the transcription factor. In both celltypes, expression was dramatically augmented by LPS (see longer exposurefor 70Z/3 cells) and, in WEH-231 cells, also by PMA, two agents that areknown to activate NF-κB in these cells. Thus gadd45β may mediate some ofthe important functions executed by NF-κB in B lymphocytes.

Example 9 The gadd45β Promoter Contains Several Putative κB Elements

To investigate the regulation of gadd45β expression by NF-κB, the muringgadd45β promoter was cloned. A BAC clone containing the gadd45β gene wasisolated from a 129SV mouse genomic library, digested with XhoI, andsubcloned into pBS vector. The 7384 bp XhoI fragment containing gadd45βwas completely sequenced, and portions were found to match sequencespreviously deposited in GeneBank (accession numbers AC073816, AC073701,and AC091518) (see also FIG. 8). The fragment harbored the genomic DNAregion spanning from ˜5.4 kbp upstream of a transcription start site tonear the end of the 4^(th) exon of gadd45β. Next, the TRANSFAC databasewas used to identify putative transcription factor-binding elements. ATATAA box was found to be located at position −56 to −60 relative to thetranscription start site (FIG. 10). The gadd45β promoter also exhibitedseveral κB elements, some of which were recently noted by others. Threestrong κB sites were found in the proximal promoter region at positions−377/−368, −426/−417, and −447/−438 (FIG. 8); whereas a weaker site waslocated as position −4516, −4890/−4881, and −5251/−5242 (FIG. 8). ThreeκB consensus sites were also noted with the first exon of gadd45β(+27/+36, +71/+80, and +171/+180). The promoter also contained an Sp1motif (−890/−881) and several putative binding sties for othertranscription factors, including heat shock factor (HSF) 1 and 2, Ets,Stat, AP1, N-Myc, MyoD, CREB, and C/EBP (FIG. 8).

To identify conserved regulatory elements, the 5.4 kbp murine DNAsequence immediately upstream of the gadd45β transcription start sitewas aligned with corresponding human sequence, previously deposited bythe Joint Genome Initiative (accession number AC005624). As shown inFIG. 8, DNA regions spanning from position −1477 to −1197 and from −466to −300 of the murine gadd45β promoter were highly similar to portionsof the human promoter (highlighted in gray are identical nucleotideswithin regions of homology), suggesting that these regions containimportant regulatory elements. A less well-conserved regions wasidentified downstream of position −183 up to the beginning of the firstintron. Additional shorter stretches of homology were also identified(see FIG. 8). No significant similarity was found upstream of position−2285. The −466/−300 homology region contained three κB sites (hereafterreferred to as κB1, κB2, and κB3), which unlike the other κB sitespresent throughout the gadd45β promoter, were conserved among the twospecies. These findings suggest that these κB sites play an importantrole in the regulation of gadd45β, perhaps accounting for the inductionof gadd45β by NF-κB.

Example 10 NF-κB Regulates the gadd45β Promoter through Three ProximalκB Elements

To determine the functional significance of the κB sites present in thegadd45β promoter, a series of CAT reporter constructs were generatedwhere CAT gene expression is driven by various portions of this promoter(FIG. 9A). Each CAT construct was transfected alone or along withincreasing amounts of RelA expression plasmids into NTera-2 embryocarcinoma cells, and CAT activity measured in cell lysates by liquidscintillation counting (FIG. 9B). RelA was chosen for these experimentsbecause of its relevance to the regulation of gadd45β expression ascompared to other NF-κB subunits (see FIG. 6). As shown in FIG. 9B, the−5407/+23-gadd45β-CATT reporter vector was dramatically transactivatedby RelA in a dose-dependent manner, exhibiting an approximately 340-foldinduction relative to the induction seen in the absence of RelA with thehighest amount of pMT2T-RelA. Qualitatively similar, RelA-dependenteffects were seen with the −3465/+23-gadd45β- and −592/+23-gadd45β-CATconstructs, which contained distal truncations of the gadd45β promoter.The relatively lower constructs, which contained distal truncations ofthe gadd45β promoter. The relatively lower basal and RelA-dependent CATactivity observed with the −5407/+23-gadd45β-CAT, may have been due, atleast in part, to the lack of a proximal 329 bp regulatory region, whichalso contained the TATA box, in the former constructs (FIGS. 9A and 9B).Even in the presence of this region, deletions extending proximally toposition −592 completely abolished the ability of RelA to activate theCAT gene (FIG. 9B, see −265/+23-gadd45β- and −103/+23-gadd45β-CATconstructs). Similar findings were obtained with analogous reporterconstructs containing an additional 116 b promoter fragment downstreamof position +23. Whereas analogously to −592/+23-gadd45β-CAT,−592/+139-gadd45β-CAT was highly response to RelA, −265/+139-gadd45β-CATwas not transactivated even by the highest amounts of pMT2T-RelA. Itshould be noted that this reporter construct failed to respond to RelAdespite retaining two putative κB binding elements at position +27/+36and +71/+80 (see FIG. 8, SEQ ID NO: 35). Together, the findings indicatethat relevant NF-κB/RelA responsive elements in the murine gadd4503promoter reside between position −592 and +23. They also imply that theκB sites contained in the first exon, as well as the distal κB sites,may not significantly contribute to the regulation of gadd45β by NF-κB.Similar conclusions were obtained in experiments employing Jurkat orHeLa cells where NF-κB was activated by PMA plus ionomycin treatment.

As shown in FIG. 8, the −592/+23 region of the gadd45(3 promotercontains three conserved κB binding sties, namely κB1, κB2, and κB3. Totest the functional significance of these κB elements, each of thesesites were mutated in the context of −592/+23-gadd45β-CAT (FIG. 10A),which contained the minimal promoter region that can be transactivatedby RelA. Mutant reporter constructs were transfected alone or along withincreasing amounts of PMT2T-RelA in NTera-2 cells and CAT activitymeasured as described for FIG. 9B. As shown in FIG. 10B, the deletion ofeach κB site significantly impaired the ability of RelA to transactivatethe −592/+23-gadd45β-CAT construct, with the most dramatic effect seenwith the mutation of κB1, resulting in a ˜70% inhibition of CAT activity(compare −592/+23-gadd45β-CAT and κB-1M-gadd45β-CAT). Of interest, thesimultaneous mutation of κB1 and κB2 impaired CAT induction byapproximately 90%, in the presence of the highest amount of transfectedRelA plasmids (FIG. 10B) (see κB-1/2M-gadd45β-CAT). Dramatic effectswere also seen when the input levels of RelA were reduced to 1 μg or 0.3μg (˜eight- and ˜five-fold reduction, respectively, as compared to thewild-type promoter). The residual CAT activity observed with the lattermutant construct was most likely due to the presence of an intact κB3site. Qualitatively similar results were obtained with the transfectionof RelA plus p50, or Rel expression constructs. It was concluded thatthe gadd45β promoter contains three functional κB elements in itsproximal region and that each is required for optimal transcriptionalactivation of NF-κB.

To determine whether these sites were sufficient to driveNF-κB-dependent transcription the Δ56-κB-1/2-, Δ56-κB-3-, andΔ56-κB-M-CAT, reporter constructs were constructed, where one copy ofthe gadd45β-κB sites or of a mutated site, respectively, were clonedinto A56-CAT to drive expression of the CAT gene (FIG. 11). Each Δ56-CATconstruct was then transfected alone or in combination with increasingamounts of RelA expression plasmids into Ntera2 cells and CAT activitymeasured as before. As shown in FIG. 11, the presence of either κB-1plus κB-2, or κB-3 dramatically enhanced the responsiveness of A56-CATto RelA. As it might have been expected from the fact that it harboredtwo, rather than one, κB sites, A56-κB-1/2-CAT was induced moreefficiently than κB3, particularly with the highest amount ofpMT2T-RelA. Low, albeit significant, RelA-dependent CAT activity wasalso noted with A56-κB-M-CAT, as well as empty A56-CAT vectors,suggesting that Δ56-CAT contains cryptic κB sites (FIG. 11). Hence,either the κB-1 plus κB-2, or κB-3 cis-acting elements are sufficient toconfer promoter responsiveness to NF-κB.

Example 11 The κB-1, κB-2, and κB-3 Elements Bind to NF-κB in vitro

To assess the ability of κB elements in the gadd45β promoter to interactwith NFκB complexes, EMSAs were performed. Oligonucleotides containingthe sequence of κB-1, κB-2, or κB-3 were radiolabeled and independentlyincubated with extracts of NTera-2 cells transfected before hand withpMT2T-p50, pMT2T-p50 plus pMT2T-RelA, or empty pMT2T plasmids, andDNA-binding complexes separated by polyacrylamide gel electrophoresis(FIG. 12A). The incubation of each κB probe with various amounts ofextract from cells expressing only p50 generated a single DNA-bindingcomplex comigrating with p50 homodimers (FIG. 12A, lanes 1-3,5-7, and9-11). Conversely, extracts from cells expressing both p50 and RelA gaverise to two specific bands: one exhibiting the same mobility of p50/p50dimers and the other comigrating with p50/RelA heterodimers (lanes 4, 8,and 12). Extracts from mock-transfected NTera2 cells did not generateany specific signal in EMSAs (FIG. 12B). Specificity of each complex wasconfirmed by competition assays where, in addition to the radiolabeledprobe, extracts were incubated with a 50-fold excess of wild-type ormutated cold κB probes. Thus, each of the functionally relevant κBelements in the gadd45β promoter can bind to NF-κB complexes in vitro.

To confirm the composition of the DNA binding complexes, supershiftassays were performed by incubating the cell extracts with polyclonalantibodies raised against human p50 or RelA. Anti-p50 antibodiescompletely supershifted the specific complex seen with extracts of cellsexpressing p50 (FIG. 12B, lanes 5, 14, and 23), as well as the twocomplexes detected with extracts of cells expressing both p50 and RelA(lanes 8, 17, and 26). Conversely, the antibody directed against RelAonly retarded migration of the slower complex seen upon concomitantexpression of p50 and RelA (lanes 9, 18, 27) and did not affect mobilityof the faster DNA-binding complex (lanes 6, 9, 15, 18, 24, and 27).

The gadd45β-κB sites exhibited apparently distinct in vitro bindingaffinities for NF-κB complexes. Indeed, with p50/RelA heterodimers, κB-2and κB-3 yielded significantly stronger signals as compared with κB-1(FIG. 12B). Conversely, κB-2 gave rise to the strongest signal with p50homodimers, whereas κB-3 appeared to associate with this complex mostpoorly in vitro (FIG. 12B). Judging from the amounts of p50/p50 andp50/RelA complexes visualized on the gel, the presence of the antibodies(especially the anti-RelA antibody) may have stabilized NF-κB-DNAinteractions (FIG. 12B). Neither antibody gave rise to any band whenincubated with the radiolabeled probe in the absence of cell extract.The specificity of the supershifted bands was further demonstrated bycompetitive binding reactions with unlabeled competitoroligonucleotides. Hence, consistent with migration patterns (FIG. 14A),the faster complex is predominantly composed of p50 homodimers, whereasthe lower one is predominantly composed of p50/RelA heterodimers. Thesedata are consistent with those obtained with CAT assays and demonstratethat each of the relevant κB elements of the gadd45, promoter canspecifically bind to p50/p50 and p50/RelA, NFκB complexes, in vitro,thereby providing the basis for the dependence of gadd45β expression onNF-κB. Hence, gadd45β is a novel direct target of NFκB.

Example 12 JNKK2 (Also Known as MKK7)-Gadd45β Interacting Domains

JNK1/2/3 are the downstream components of one of the majormitogen-activated protein kinase (MAPK) cascades, also comprising theextracellular signal-regulated kinase (ERK1/2) and p38(α/β/γ/δ)cascades. MAPKs are activated by MAPK kinases (MAPKKs), which in turnare activated by MAPKK kinases (MAPKKKs). To understand the basis forthe Gadd45β control of JNK signaling was determined whether Gadd45βcould physically interact with kinases in these cascades. HA-taggedkinases were transiently expressed in 293 cells, alone or together withFLAG-Gadd45β, and associations were assessed by combinedimmunoprecipitation and Western blot assays. Gadd45β bound to ASK1, butnot to other MAPKKKs capable of interacting with TRAF2 (FIG. 26 a,left), a factor required for JNK activation by TNFα. It also associatedwith MEKK4/MTK1−a MAPKKK that instead is not induced by TNFα. Notably,Gadd45β interacted strongly with MKK7/JNKK2, but not with the other JNKkinase, MKK4/JNKK1, the p38-specific activators MKK3b and MKK6, or theERK kinase, MEK-1, as well as with MAPKs (FIG. 26 a, middle and right,and FIG. 26 b). Gadd45β interactions were confirmed in vitro.Glutathione S-transferase (GST)-Gadd45β, but not GST, precipitated alarge fraction of the MKK7 input (FIG. 26 c), whereas it absorbed only asmall fraction of ASK1 or MEKK4. Hence, Gadd45β interacts withJNK-inducing kinases and most avidly with MKK7.

Another question was whether Gadd45β association with these kinases hadfunctional consequences, in vivo. Remarkably, whereas in IκBαM-Hygro 3DOcontrol clones, TNFα activated MKK7 strongly, in clones expressingGadd45β this activation was abolished (FIG. 27 a). Inhibition wasspecific since Gadd45, had no effect on induction of other MAPKKs (i.e.MKK4, MKK3/6, and MEK1/2) by either TNFα or PMA plus ionomycin (P/I;FIG. 27 b and FIG. 27 c, respectively). ASK1 and MEKK1 were activatedweakly by TNFα, and this activation too was unaffected by Gadd45β (FIG.27 b). Thus, Gadd45β selectively blocked induction of MKK7phosphorylation/activity by TNFα.

Gadd45β mediates the suppression of JNK signaling by NF-κB. Indeed, MKK7was inhibited by NF-κB (FIG. 27 d). Whereas in control 3DO clones (Neo),MKK7 activation by TNFα returned to basal levels by 40 minutes—therebymirroring the JNK response—in NF-κB-null clones (IκBαM), this activationremained sustained. MKK7 down-regulation correlated with Gadd45βinduction by NF-κB. Furthermore, NF-κB did not affect MKK4, MKK3/6, orMEK1/2 (FIG. 27 d and FIG. 27 e), thereby recapitulating the effects ofGadd45β on MAPK cascades.

Interaction of endogenous Gadd45β and MKK7 was detected readily (FIG. 28a). Anti-Gadd45β monoclonal antibodies co-immunoprecipitated MKK7 fromP/I-treated 3DO cells, exhibiting strong Gadd45β expression (bottomright), but not from untreated cells, lacking detectable Gadd45β. MKK7was present at comparable levels in stimulated and unstimulated cells(bottom, left) and was not co-precipitated by an isotype-matched controlantibody. The interaction was confirmed by using anti-MKK7 antibodiesfor immunoprecipitation and the anti-Gadd45β monoclonal antibody forWestern blots (FIG. 28 a, top right). Anti-MEKK1 antibodies failed toco-precipitate Gadd45β, further demonstrating the specificity of theMKK7-Gadd45β association. To determine whether Gadd45β binds to MKK7directly, we used purified proteins (FIG. 28 b). Purified GST-MKK7 orGST were incubated, in vitro, with increasing amounts of purifiedHis₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46) or control His₆-JIP1(His₆ disclosed as SEQ ID NO: 46), and the fraction of His₆-taggedpolypeptides (His₆ disclosed as SEQ ID NO: 46) that bound to GSTproteins was visualized by Western blotting. His₆-Gadd45β (His₆disclosed as SEQ ID NO: 46) specifically associated with GST-MKK7 (FIG.28 c), and this association was tighter than that of the physiologicMKK7 regulator, JIP1, with the half maximum binding (HMB) values being˜390 nM for Gadd45β and above 650 nM for JIP1 (left; JIP1 was used undernon-saturating conditions). Endogenous Gadd45β and MKK7 likely associatevia direct, high-affinity contact.

A question was whether Gadd45β inhibited active MKK7, in vitro.FLAG-MKK7 was immunoprecipitated from TNFα-treated or untreated 293cells, and kinase assays were performed in the presence of purifiedHis₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46), GST-Gadd45β, or controlproteins (FIG. 28 d; see also FIG. 28 g). Both Gadd45β polypeptides, butneither GST nor His₆-EF3 (His₆ disclosed as SEQ ID NO: 46), blockedGST-JNK1 phosphorylation by MKK7, in a dose-dependent manner (FIG. 28d). Consistent with the in vivo findings (FIG. 27), the inhibitoryactivity of Gadd45β was specific. In fact, even at high concentrations,this factor did not hamper MKK4, MKK3b, or—despite its ability to bindto it in over-expression (FIG. 26 a)—ASK1 (FIG. 28 e; see also FIG. 28f, total levels). Hence, Gadd45β is a potent and specific inhibitor ofMKK7. Indeed, the effects of Gadd45β on MKK7 phosphorylation by TNFα maybe due inhibition of the MKK7 ability to auto-phoshorylate and/or toserve as substrate for upstream kinases. Altogether, the findingsidentify MKK7 as a target of Gadd45β, and of NF-□B, in the JNK cascade.Of interest, MKK7 is a selective activator of JNK, and its ablationabolishes JNK activation by TNFα. Thus, blockade of MKK7 is sufficienton its own to explain the effects of Gadd45β on JNK signaling—i.e. itsspecific and near-complete suppression of this signaling.

The amino acid sequence of Gadd45β is not similar to sequences ofphosphatases and is not known to have enzymatic activity. Thus, tounderstand mechanisms of kinase inactivation, the Gadd45β-bindingregion(s) of MKK7 were mapped using sets of N- and C-terminallytruncated MKK7 polypeptides (FIG. 29 a and FIG. 29 c, respectively).Full length nucleotide and amino acid sequences of human and murine MKK7or JNKK2 are shown in FIG. 31. As used herein, the amino acid positionsrefer to a human MKK7 or JNKK2 amino acid sequence. MKK7/63-401,MKK7/91-401, and MKK7/132-401 bound to GST-Gadd45β specifically and withaffinity comparable to that of full-length MKK7, whereas mutationsoccurring between amino acids 157 and 213 interacted weakly withGST-Gadd45β (FIG. 29 b). Ablation of a region extending to or beyondresidue 232 abolished binding. Analysis of C-terminal truncationsconfirmed the presence of a Gadd45β-interaction domain between residues141 and 161 (FIG. 29 d; compare MKK7/1-140 and MKK7/1-161), but failedto reveal the C-terminal binding region identified above, suggestingthat Gadd45β interacts with this latter region more weakly. Hence, MKK7contacts Gadd45β through two distinct regions located within residues132-161 and 213-231 (hereafter referred to as region A and B,respectively).

To define interaction regions and determine whether they are sufficientfor binding, Gadd45β association with overlapping peptides spanningthese regions (FIG. 29 e) was determined. As shown in FIG. 29 f, bothregions A and B bound to GST-Gadd45β—even when isolated from the contextof MKK7—and peptides 132-156 and 220-234 (i.e. peptides 1 and 7,respectively) were sufficient to recapitulate this binding. Bothpeptides lie within the MKK7 kinase domain, and peptide 1 spans theATP-binding site, K149, required for catalytic function—suggesting thatGadd45, inactivates MKK7 by masking critical residues. This isreminiscent of the mechanism by which p27^(KIP1) inhibitscyclin-dependent kinase (CDK)2. A question explored was whether MKK7,Gadd45β-binding peptides interfered with the Gadd45β ability to suppresskinase activity. Indeed, peptide 1 prevented MKK7 inhibition by Gadd45β,whereas peptide 7 or control peptides did not (FIG. 30 a). Hence, kinaseinactivation by Gadd45β requires contact with region A, but not withregion B.

These data predict that preventing MKK7 inactivation by Gadd45β, invivo, should sensitize cells to TNFα-induced apoptosis. To test thishypothesis, MKK7-mimicking peptides were fused to a cell-permeable,HIV-TAT peptide and transduced into cells. Remarkably, peptide 1markedly increased susceptibility of IκBαM-Gadd45β cells to TNFα-inducedkilling, whereas DMSO-treated cells were resistant to this killing, asexpected (FIG. 30 b). Importantly, peptide 1 exhibited marginal basaltoxicity, indicating that its effects were specific for TNFαstimulation, and other peptides, including peptide 7, had no effect onthe apoptotic response to TNFα. Consistent with the notion that MKK7 isa target of NF-κB, peptide 1 promoted TNFα-induced killing inNF-κB-proficient cells (Neo; FIG. 30 c)—which are normally refractory tothis killing. As seen with Gadd45β-expressing clones, this peptideexhibited minimal toxicity in untreated cells. Together, the findingssupport that Gadd45β halts the JNK cascade by inhibiting MKK7 andcausally link the Gadd45β protective activity to this inhibition.Furthermore, blockade of MKK7 is a factor in the suppression ofapoptosis by NF-κB, and this blockade is mediated, at least in part, byinduction of Gadd45 β.

A mechanism for the control of JNK signaling by Gadd45β was identified.Gadd45β associates tightly with MKK7, inhibits its enzymatic activity bycontacting critical residues in the catalytic domain, and thisinhibition is a factor in its suppression of TNFα-induced apoptosis.Interactions with other kinases do not appear relevant to the Gadd45,control of JNK activation and PCD by TNFα, because MEKK4 is not involvedin TNF-R signaling, and ASK1 is apparently unaffected by Gadd45β.Indeed, peptides that interfere with Gadd45β binding to MKK7 blunt theGadd45, protective activity against TNFα (FIG. 30 a and FIG. 30 b). Thetargeting of MKK7 is a factor in the suppression of apoptosis by NF-κB.NF-κB-deficient cells fail to down-modulate MKK7 induction by TNFα, andMKK7-mimicking peptides can hinder the ability of NF-κB to blockcytokine-induced killing (FIG. 30 c). These results appear consistentwith a model whereby NF-κB activation induces transcription of Gadd45β,which in turn inhibits MKK7, leading to the suppression of JNKsignaling, and ultimately, apoptosis triggered by TNFα.

Chronic inflammatory conditions such as rheumatoid arthritis andinflammatory bowel disease are driven by a positive feedback loopcreated by mutual activation of TNFα and NF-κB. Furthermore, severalmalignancies depend on NF-κB for their survival—a process that mightinvolve suppression of JNK signaling. These results suggest thatblockade of the NF-κB ability to shut down MKK7 may promote apoptosis ofself-reactive/pro-inflammatory cells and, perhaps, cancer cells, therebyidentifying the MKK7-Gadd45β interaction as a potential therapeutictarget. Interestingly, pharmacological compounds that disrupt Gadd45βbinding to MKK7 might uncouple anti-apoptotic and pro-inflammatoryfunctions of NF-κB, and so, circumvent the potent immunosuppressiveside-effects seen with global NF-κB blockers—currently used to treatthese illnesses. The pro-apoptotic activity of MKK7 peptides inNF-κB-proficient cells implies that, even if NF-κB were to induceadditional MKK7 inhibitors, these inhibitors would target MKK7 throughits Gadd45β-binding surface, thereby proving in principle the validityof this therapeutic approach.

Example 13 MKK7 Inactivation by Gadd45β In Vivo, Sensitizes Cells toTNFα-Induced Apoptosis

NF-κB/Rel transcription factors regulate apoptosis or programmed celldeath (PCD), and this regulation plays a role in oncogenesis, cancerchemo-resistance, and to antagonize tumor necrosis factor (TNF)α-inducedkilling. Upon TNFα induction, the anti-apoptotic activity of NF-κBinvolves suppressing the c-Jun-N-terminal kinase (JNK) cascade.Gadd45β/Myd118, a member of the Gadd45 family of inducible factors playsan important role in this suppressive activity of NF-κB. However, themechanisms by which Gadd45β blunts JNK signaling are not understood.MKK7/JNKK2 is identified as a specific and an essential activator of JNKsignaling and as a target of Gadd45β and also NF-κB itself. Gadd45βbinds to MKK7 directly and blocks its catalytic activity, therebyproviding a molecular link between the NF-κB and JNK pathways. Gadd45βis required to antagonize TNFα-induced cytotoxicity, and peptidesdisrupting the Gadd45β/MKK7 interaction hinder the ability of Gadd45β,as well as of NF-κB, to suppress this cytotoxicity. These resultsestablish a basis for the NF-κB control of JNK activation and identifyMKK7 as a potential target for anti-inflammatory and anti-cancertherapy.

These data predict that preventing. MKK7 inactivation by Gadd45β, invivo, sensitizes cells to TNFα-induced apoptosis. MKK7-mimickingpeptides were fused to a cell-permeable, HIV-TAT peptide and transducedinto cells. As shown by flow cytometry (FCM) and confocal microscopy,peptides entered cells with equivalent efficiency (FIG. 34 a-d). Peptide1 markedly increased susceptibility of IκBαM-Gadd45β cells toTNFα-induced killing, whereas DMSO-treated cells were resistant to thiskilling, as expected (FIG. 33 a, left;). Peptide 1 exhibited marginalbasal toxicity indicating that its effects were specific for TNFαstimulation, and other peptides, including peptide 7, had no effect onthe apoptotic response to TNFα. Further linking the in vivo effects ofpeptide 1 to Gadd45β, pro-apoptotic activity of Ala mutant peptidescorrelated with their apparent binding affinity for Gadd45β, in vitro(FIGS. 32 d and 33 a, right). Consistent with the notion that MKK7 is atarget of NF-κB, peptide 1 promoted TNFα-induced killing inNF-κB-proficient cells (Neo; FIG. 33 b)—which are normally refractory tothis killing. As seen with Gadd45β-expressing clones, this peptideexhibited minimal toxicity in untreated cells, and mutation of residuesrequired for interaction with Gadd45β abolished its effects on TNFαcytotoxicity (FIG. 33 b, right). Together, the findings demonstrate thatGadd45β halts the JNK cascade by inhibiting MKK7 and causally link theGadd45βprotective activity to this inhibition. Furthermore, blockade ofMKK7 is crucial to the suppression of apoptosis by NF-κB, and thisblockade is mediated, at least in part, by induction of Gadd45β.

Chronic inflammatory conditions such as rheumatoid arthritis andinflammatory bowel disease are driven by a positive feedback loopcreated by mutual activation of TNFα and NF-κB. Furthermore, severalmalignancies depend on NF-κB for their survival—a process that mightinvolve the suppression of JNK signaling. The results suggest thatblockade of the NF-κB ability to shut down MKK7 may promote apoptosis ofself-reactive/pro-inflammatory cells and, perhaps, of cancer cells,thereby identifying the MKK7-Gadd45β interaction as a potentialtherapeutic target. Pharmacological compounds that disrupt Gadd45,binding to MKK7 might uncouple anti-apoptotic and pro-inflammatoryfunctions of NF-κB, and so, circumvent the potent immunosuppressiveside-effects seen with global NF-κB blockers-currently used to treatthese illnesses. The pro-apoptotic activity of MKK7 peptides inNF-κB-proficient cells indicates that critical NF-κB-inducibleinhibitors target MKK7 through or in vicinity of its Gadd45β-bindingsurface, thereby proving in principle the validity of this therapeuticapproach.

Example 14 Cell-Specific Modulation of JNKK2 Activity

In mouse embryonic fibroblasts (MEFs), Gadd45β ablation was reported notto affect TNFα-induced PCD. The effects of MKK7-derived peptides weretested in these cells. The peptide 2 (aa 142-166 of MKK7/JNKK2) has anamino acid sequence NH2-TGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 1)and the TAT fusion version has an amino acid sequence NH2-GRKKRRQRRRPPTGHVIAVKQMRRSGNKEENKRILMD-COOH (SEQ ID NO: 45).

FIGS. 33A-B shows that the Gadd45β-mediated suppression of MKK7 isrequired to block TNFα-induced apoptosis. This is shown by the findingthat MKK7-mimicing peptide 1, which prevents the Gadd45β-mediatedinhibition of MKK7, sensitizes IκBαM-Gadd45β (FIG. 33A) and Neo (FIG.33B) 3DO clones, respectively, to TNFα-induced apoptosis. MKK7-mimickingpeptides were fused to a cell-permeable, HIV-TAT peptide and transducedinto cells. As shown in FIG. 34, peptides entered cells with equivalentefficiency. Remarkably, peptide 1 markedly increased susceptibility ofIκBαM-Gadd45β cells to TNFα-induced killing, whereas DMSO-treated cellswere resistant to this killing (FIG. 33A, left; see also FIG. 35A), asexpected (De Smaele et al., 2001). Other peptides, including peptide 7,had no effect on the apoptotic response to TNFα. Peptide 1 exhibitedmarginal basal toxicity (FIG. 35A, left) indicating that its effect wasspecific for cytokine stimulation. Further linking the in vivo effect ofpeptide 1 to Gadd45β, pro-apoptotic activity of Ala mutant peptidescorrelated with their apparent binding affinity for Gadd45, in vitro(FIG. 32).

FIG. 33B shows that, consistent with the notion that MKK7 is a target ofNF-κB, peptide 1 promoted TNFα-induced killing in NF-κB-proficient cells(Neo; FIG. 33B; see also FIG. 35B)—which are expected to be refractoryto this killing (De Smaele et al., 2001). As seen withGadd45β-expressing clones, this peptide exhibited minimal toxicity inuntreated cells (FIG. 35B, left), and mutation of residues required forinteraction with Gadd45β abolished its effects on TNFα cytotoxicity(FIG. 33B, right). Together, the findings demonstrate that Gadd45β haltsthe JNK cascade by inhibiting MKK7 and causally links the Gadd45βprotective activity to this inhibition. Furthermore, blockade of MKK7 iscrucial to the suppression of apoptosis by NF-κB, and this blockade ismediated, at least in part, by induction of Gadd45β.

FIG. 33C-D depicts apoptosis assays showing that both peptide 1 andpeptide 2 facilitate TNFα-induced killing in wild-type MEFs, and thatonly peptide 2 promotes this killing in Gadd45β null MEFs, respectively.MEFs were from twin embryos and were used at passage (p)₄. This figureshows that Gadd45β is required to block MKK7 activation and apoptosisinduction by TNFα. It also shows that in some cell types (e.g.fibroblasts), at least another factor, distinct from Gadd45β, isessential to execute these functions. A recent report suggested that, inmouse embryonic fibroblasts (MEFs), Gadd45β ablation does not affectTNFα-induced PCD (Amanullah et al., 2003). The effects of MKK7-derivedpeptides were tested in these cells. Surprisingly, in wild-typefibroblasts cytokine-induced toxicity was enhanced by both peptide 1 andpeptide 2, whereas other peptides had no effect on this toxicity (FIG.33C, see also FIG. 35C). This contrasts with what was seen in 3DOlymphoid cells, where only peptide 1 promoted killing by TNFα (FIG.33B). Because peptide 2 does not bind to Gadd45β (FIG. 29), itspro-apoptotic activity is most likely due to displacement of anotherinhibitory factor(s) from MKK7.

Consistent with this notion, activity of peptide 2 was retained (and, infact, enhanced) in gadd45β^(−/−), MEFs (FIG. 33D; see also FIG. 35D).Remarkably, however, Gadd45β ablation rendered these cells completelyinsensitive to the cytotoxic effects of peptide 1 (FIGS. 33D and 35D),indicating that in wild-type fibroblasts, these effects were due toGadd45β inactivation. Together, these findings demonstrate that the MKK7inhibitory mechanism activated in response to TNFα is tissue-specific(shown by the distinct effects of MKK7 peptides in 3DO cells andfibroblasts; FIGS. 33B-D), and that, at least in MEFs, this mechanism isfunctionally redundant. They also provide compelling evidence thatGadd45β is required to antagonize TNFα-induced killing (FIG. 35C).Indeed, the apparent lack of apoptotic phenotype previously reported ingadd45β^(−/−) fibroblasts (Amanullah et al., 2003) appears due toactivation of compensatory mechanisms in these cells—mechanisms that arenot mounted during acute Gadd45β inactivation by peptide 1.

A mechanism for the control of JNK signaling by Gadd45β is identified.Gadd45β associates tightly with MKK7, inhibits its enzymatic activity bycontacting critical residues in the catalytic domain, and thisinhibition is crucial to the suppression of TNFα-induced apoptosis.Interactions with other kinases do not appear relevant to the Gadd45βcontrol of JNK activation and PCD by TNFα, as MEKK4 is not involved inTNF-R signaling, and ASK1 is seemingly unaffected by Gadd45β (FIGS.21-22). Indeed, peptides that interfere with Gadd45β binding to MKK7blunt the Gadd45β protective activity against TNFα (FIGS. 33A, 33C, 33D,35A, 35C, 35D). The targeting of MKK7 effects suppression of apoptosisby NF-κB itself. NF-κB-deficient cells fail to down-modulate MKK7induction by TNFα, and MKK7-mimicking peptides disrupting theGadd45β/MKK7 interaction hinder the ability of NF-κB to blockTNFα-induced cytotoxicity (FIGS. 33B-C). A model is that NF-κBactivation induces expression of Gadd45β, which in turn inhibits MKK7,leading to the suppression of JNK signaling, and ultimately, apoptosistriggered by TNFα. These findings identify a molecular link between theNF-κB and JNK pathways, and establish a basis for the NF-κB control ofJNK activation. Indeed, the relevance of this link is underscored byknockout studies showing that Gadd45β is essential to antagonizeTNFα-induced apoptosis (FIGS. 33B-C). Yet, in some tissues, otherNF-κB-inducible factors might contribute to suppress MKK7 induction byTNFα (FIGS. 33B-C).

Chronic inflammatory conditions such as rheumatoid arthritis andinflammatory bowel disease are driven by a positive feedback loopcreated by mutual activation of TNFα and NF-κB. Furthermore, severalmalignancies depend on NF-κB for their survival—a process that mightinvolve suppression of JNK signaling. Blockade of the NF-κB ability toshut down MKK7 may promote apoptosis of self-reactive/pro-inflammatorycells and, perhaps, of cancer cells, thereby identifying theMKK7-Gadd45β interaction as a potential therapeutic target.Pharmacological compounds that disrupt Gadd45β binding to MKK7 mightuncouple anti-apoptotic and pro-inflammatory functions of NF-κB, and so,circumvent the potent immunosuppressive side-effects seen with globalNF-κB blockers—currently used to treat these illnesses. Thepro-apoptotic activity of MKK7 peptides in NF-κB-proficient cellsimplies that NF-κB-inducible factors target MKK7 through or in proximityof its Gadd45β-binding surface, thereby proving in principle thevalidity of this therapeutic approach.

Example 15 Regions of Gadd45β that Bind to and Inhibit MKK7

FIG. 36 shows that the 69-86 amino acid region of Gadd45β is sufficientto bind to MKK7 in vitro. GST pull-down assays were performed using GST-or GST-MKK7-coated beads and in vitro-translated, Gadd45β productscorresponding to the polypeptidic fragments indicated in FIG. 36A.

FIG. 37 shows that the Gadd45β-mediated inhibition of MKK7 requires apolypeptidic region of Gadd45β including the region between amino acid60 and 86. Active MKK7 was immunoprecipitated from TNFα-activated 293cells and MKK7 kinase assays were performed using GST-JNK1 substratesand pure recombinant Gadd45β polypeptides (FIG. 37B; a schematic diagramrepresenting the Gadd45βpolypeptides used is shown in FIG. 37A). FIGS.37D-E show that the amino acid regions contained in the overlapping,Gadd45β-derived peptides 2 and 8 are sufficient to recapitulate most ofthe inhibitory activity of Gadd45β on MKK7. MKK7 kinase assays wereperformed as in FIG. 37B, except that pure synthetic Gadd45, peptides(whose sequences are shown in FIG. 37C) were used instead of purerecombinant Gadd45β proteins. The amino acid region between amino acids58 and 77 of Gadd45βis used for the Gadd45β-mediated inhibition of MKK7.Thus, it is expected that cell-permeable forms of these peptides can beused in cells to block apoptosis induced by TNFα or other pro-apoptoticagents. These peptides could also used in the whole animal to blockapoptosis in inflammatory diseases, neurodegenerative disorders, stroke,and myocardial infarction.

Materials and Methods 1. Library Preparation and Enrichment

cDNA was prepared from TNFα-treated NIH-3T3 cells and directionallyinserted into the pLTP vector (Vito et al., 1996). For the enrichment,RelA−/− cells were seeded into 1.5×10⁶/plate in 100 mm plates and 24hours later used for transfection by of the spheroplasts fusion method.A total of 4.5×10⁶ library clones were transfected for the first cycle.After a 21-hours treatment with TNFα (100 units/ml) and CHX (0.25μg/ml), adherent cells were harvested for the extraction of episomal DNAand lysed in 10 mM EDTA, 0.6% SDS for the extraction of episomal DNAafter amplification, the library was used for the next cycle ofselection. A total of 4 cycles were completed.

2. Constructs

IκBαM was excised from pCMX-IκBαM (Van Antwerp et al., 1996) and ligatedinto the EcoRI site of pcDNA3-Neo (Invitrogen). Full length human RelAwas PCR-amplified from BS-RelA (Franzoso et al., 1992) and inserted intothe BamHI site of pEGFP-C1 (Clontech). Gadd45β, Gadd45α and Gadd45γcDNAs were amplified by PCR for the pLTP library and cloned into theXhoI site and pcDNA 3.1-Hygro (Invitrogen) in both orientations. Togenerate pEGFP-Gadd45β, Gadd45β was excised from pcDNA Hygro withXhoI-XbaI and ligated with the linker5′-CTAGAGGAACGCGGAAGTGGTGGAAGTGGTGGA-3′ (SEQ ID NO: 13) into theXbaI-BamHI sites of pEGFP-N1. pcDNA-Gadd45α was digested with EcoRI-XhoIand ligated with XhoI-BamHI opened pEGFP-C1 and the linker5′-GTACAAGGGAAGTGGTGGAAGTGTGGAATGACTTTGGAGG-3′ (SEQ ID NO: 14).pEGFP-N-1-Gadd45γ was generated by introducing the BspEI-XhoI fragmentof pcDNA-Hygro-Gadd45γ along with the adapter5′-ATTGCGTGGCCAGGATACAGTT-3′ (SEQ ID NO: 15) into pEGFP-C1-Gadd45α,where Gadd45α was excised by EcoRI-SalI. All constructs were checked bysequencing. pSRα3 plasmids expressing DN-JNKK1 (S257A, T261A), DN-JNKK2(K149M, S271A, T275A) and MKK3bDN (S128A, T222A) were previouslydescribed (Lin et al., 1995; Huang et al., 1997).

3. Anti Sense Constructs of gadd45β

Modulators of the JNK pathway, such as Gadd45β, can be modulated bymolecules that directly affect RNA transcripts encoding the respectivefunctional polypeptide. Antisense and ribozyme molecules are examples ofsuch inhibitors that target a particular sequence to achieve areduction, elimination or inhibition of a particular polypeptide, suchas a Gadd45 sequence or fragments thereof.

Antisense methodology takes advantage of the fact that nucleic acidstend to pair with “complementary” sequences. Antisense constructsspecifically form a part of the current invention, for example, in orderto modulate the JNK pathway. In one embodiment of the invention,antisense constructs comprising a Gadd45 nucleic acid are envisioned, inantisense orientation, as well as portions of fragments thereof.

By complementary, it is meant that polynucleotides are those which arecapable of base-pairing according to the standard Watson-Crickcomplementarity rules. That is, the larger purines will base pair withthe smaller pyrimidines to form combinations of guanine paired withcytosine (G:C) and adenine paired with either thymine (A:T) in the caseof DNA, or adenine paired with uracil (A:U) in the case of RNA.Inclusion of less common bases such as inosine, 5-methylcytosine,6-methyladenine, hypoxanthine and others in hybridizing sequences doenot interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNAs, may be employed to inhibit gene transcription or translation ofboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs, including synthetic anti-sense oligonucleotides,may be designed to bind to the promoter and other control regions,exons, introns or even exon-intron boundaries of a gene. It iscontemplated that the most effective antisense constructs may includeregions complementary to intron/exon splice junctions. Thus, antisenseconstructs with complementarily to regions within 50-200 bases of anintron-exon splice junction may be used. It has been observed that someexon sequences can be included in the construct without seriouslyaffecting the target selectivity thereof. The amount of exonic materialincluded will vary depending on the particular exon and intron sequencesused. One can readily test whether too much exon DNA is included simplyby testing the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to generate specific constructs. For example, wherean intron is desired in the ultimate construct, a genomic clone willneed to be used. The cDNA or a synthesized polynucleotide may providemore convenient restriction sites for the remaining portion of theconstruct and, therefore, would be used for the rest of the sequence.

4. Cell Lines, Transfections and Treatments

MEF and 3DO cells were cultured in 10% Fetal bovine serum-supplementedDMEM and RPMI, respectively. Transient transfections in RelA−/− MEF wereperformed by Superfect according to the manufacturer's instructions(Qiagen). After cytotoxic treatment with CHX (Sigma) plus or minus TNFα(Peprotech), adherent cells were counted and analyzed by FCM (FACSort,Becton Dickinson) to assess numbers of live GFP⁺ cells. To generate 3DOstable lines, transfections were carried out by electroporatoration(BTX) and clones were grown in appropriate selection media containingGeneticin (Gibco) and/or Hygromycin (Invitrogen). For the assessment ofapoptosis, 2DO cells were stained with PI (Sigma) and analyzed by FCM,as previously described (Nicoletti et al., 1991). Daunorubicin, PMA,Ionomycin, hydrogen peroxide, and sorbitol were from Sigma; Cisplatin(platinol AQ) was from VHAplus, PD98059 and SB202190 were fromCalbiochem.

5. Northern Blots, Western Blots, EMSAs, and Kinase Assays

Northern blots were performed by standard procedures using 6 μg of totalRNA. The EMSAs with the palindromic probes and the preparation of wholecell extracts were as previously described (Franzoso et al., 1992). Forwestern blots, cell extracts were prepared either in a modified lysisbuffer (50 mM Tris, pH 7.4, 100 mM NaCl, 50 mM NaF, 1 mM NaBo₄, 30 mMpyrophosphate, 0.5% NP-40, and protease inhibitors (FIG. 1B; BoehringerMannheim), in Triton X-100 buffer (FIG. 4A; Medema et al., 1997) or in alysis buffer containing 1% NP-40 350 mM NaCl, 20 MM HEPES (pH 8.0), 20%glycerol, 1 mM MgCl₂, 0.1 mM EGTA, 0.5 mM DTT, 1 mM Na₃VO₄, 50 mM NaFand protease inhibitors. Each time, equal amounts of proteins (rangingbetween 15 and 50 μg) were loaded and Western blots prepared accordingto standard procedures. Reactions were visualized by ECL (Amersham).Antibodies were as follows: IκBα, Bid, and β-actin from Santa CruzBiotechnology; caspase-6, -7 and -9, phospho and total -p38, phosph andtotal -ERK, phospho and total -JNK from Cell Signaling Technology;caspase-8 from Alexis; Caspase-2 and -3 from R&D systems. TheGadd45β-specific antibody was generated against an N-Terminal peptide.Kinase assays were performed with recombinant GST-c-jun and anti-JNKantibodies (Pharmingen), (Lin et al., 1995).

6. Measurement of Caspase Activity and Mitochondrial TransmembranePotential

For caspase in vitro assays, cells were lysed in Triton X-100 buffer andlysates incubated in 40 μM of the following amino trifluromethylcoumarin (ATC)-labeled caspase-specific peptides (Bachem): xVDVAD(caspase 2), zDEVD (caspases 3/7), xVEID (caspase 6), xIETD (caspase 8),and Ac-LEHD (caspase 9). Assays were carried out as previously described(Stegh et al., 2000) and specific activities were determined using afluorescence plate reader. Mitochondrial transmembrane potential wasmeasured by means of the fluorescent dye JC-1 (Molecular Probes, Inc.)as previously described (Scaffidi et al., 1999). After TNFα treatment,cells were incubated with 1.25 μg/ml of the dye for 10 min at 37° C. inthe dark, washed once with PBS and analyzed by FCM.

7. Therapeutic Application of the Invention

The current invention provides methods and compositions for themodulation of the JNK pathway, and thereby, apoptosis. In one embodimentof the invention, the modulation can be carried out by modulation ofGadd45β and other Gadd45 proteins or genes. Alternatively, therapy maybe directed to another component of the JNK pathway, for example, JNK1,JNK2, JNK3, MAPKKK (Mitogen Activated Protein Kinase Kinase Kinase):GCK, GCKR, ASK1/MAPKKK5, ASK2/MAPKKK6, DLK/MUK/ZPK, LZK, MEKK1, MEKK2,MEKK3, MEKK4/MTK1, MLK1, MLK2/MST, MLK3/SPRK/PTK1, TAK1, Tpl-2/Cot.MAPKK (Mitogen Activated Protein Kinase Kinase):MKK4/SEK1/SERK1/SKK1/JNKK1, MKK7/SEK2/SKK4/JNKK2. MAPK (MitogenActivated Kinase): JNK1/SAPKγ/SAPK1c, JNK2/SAPKα/SAPK1a,JNK3/SAPKβ/SAPK1b/p49F12.

Further, there are numerous phosphatases, scaffold proteins, includingJIP1/IB1, JIP2/IB2, JIP3/JSAP and other activating and inhibitorycofactors, which are also important in modulating JNK signaling and maybe modulated in accordance with the invention. Therapeutic uses aresuitable for potentially any condition that can be affected by anincrease or decrease in apoptosis. The invention is significant becausemany diseases are associated with an inhibition or increase ofapoptosis. Conditions that are associated with an inhibition ofapoptosis include cancer; autoimmune disorders such as systemic lupuserythemaosus and immune-mediated glomerulonephritis; and viralinfections such as Herpesviruses, Poxviruses and Adenoviruses. Theinvention therefore provides therapies to treat these, and otherconditions associated with the inhibition of apoptosis, which compriseadministration of a JNK pathway modulator that increases apoptosis. Asupregulation of Gadd45 blocks apoptosis, diseases caused by inhibitionof apoptosis will benefit from therapies aimed to increase JNKactivation, for example via inhibition of Gadd45. one example of a waysuch inhibition could be achieved is by administration of an antisenseGadd45 nucleic acid.

Particular uses for the modulation of apoptosis, and particularly theincrease of apoptosis, are for the treatment of cancer. In theseinstances, treatments comprising a combination of one or more othertherapies may be desired. For example, a modulator of the JNK pathwaymight be highly beneficial when used in combination with conventionalchemo- or radio-therapies. A wide variety of cancer therapies, known toone of skill in the art, may be used individually or in combination withthe modulators of the JNK pathway provided herein. Combination therapycan be used in order to increase the effectiveness of a therapy using anagent capable of modulating a gene or protein involved in the JNKpathway. Such modulators of the JNK pathway may include sense orantisense nucleic acids.

One example of a combination therapy is radiation therapy followed bygene therapy with a nucleic acid sequence of a protein capable ofmodulating the JNK pathway, such as a sense or antisense Gadd45β nucleicacid sequence. Alternatively, one can use the JNK modulator basedanti-cancer therapy in conjunction with surgery and/or chemotherapy,and/or immunotherapy, and/or other gene therapy, and/or local heattherapy. Thus, one can use one or several of the standard cancertherapies existing in the art in addition with the JNK modulator-basedtherapies of the present invention.

The other cancer therapy may precede or follow a JNK pathwaymodulator-based therapy by intervals ranging from minutes to days toweeks. In embodiments where other cancer therapy and a Gadd45βinhibitor-based therapy are administered together, one would generallyensure that a significant period of time did not expire between the timeof each delivery. In such instances, it is contemplated that one wouldadminister to a patient both modalities without about 12-24 hours ofeach other and, more preferably, within about 6-12 hours of each other,with a delay time of only about 12 hours being most preferred. In somesituations, it may be desirable to extend the time period for treatmentsignificantly, however, where several days (2, 3, 4, 5, 6 or 7) toseveral weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respectiveadministrations.

It also is conceivable that more than one administration of eitheranother cancer therapy and a Gadd45β inhibitor-based therapy will berequired to achieve complete cancer cure. Various combinations may beemployed, where the other cancer therapy is “A” and a JNK pathwaymodulator-based therapy treatment, including treatment with a Gadd45inhibitor, is “B”, as exemplified below:

-   -   A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B A/A/B/B        A/B/A/B A/B/B/A B/B/A/A/ B/AB/A B/A/A/B B/B/B/A A/A/A/B B/A/A/A        A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B

Other combinations also are contemplated. A description of some commontherapeutic agents is provided below.

8. Chemotherapeutic Agents

In the case of cancer treatments, another class of agents for use incombination therapy are chemotherapeutic agents. These agents arecapable of selectively and deleteriously affecting tumor cells. Agentsthat cause DNA damage comprise one type of chemotherapeutic agents. Forexample, agents that directly cross-link DNA, agents that intercalateinto DNA, and agents that lead to chromosomal and mitotic aberrations byaffecting nucleic acid synthesis. Some examples of chemotherapeuticagents include antibiotic chemotherapeutics such as Doxorubicin,Daunorubucin, Mitomycin (also known as mutamycin and/or mitomycin-C),Actinomycine D (Dactinomycine), Bleomycin, Plicomycin. Plant alkaloidssuch as Taxol, Vincristine, Vinblastine. Miscellaneous agents such asCisplatin, VP16, Tumor Necrosis Factor. Alkylating Agents such as,Carmustine, Melphalan (also known as alkeran, L-phenylalanine mustard,phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalaninederivative of nitrogen mustard), Cyclophosphamide, Chlorambucil,Busulfan (also known as myleran), Lomustine. And other agents forexample, Cisplatin (CDDP), Carboplatin, Procarbazine, Mechlorethamine,Camptothecin, Ifosfamide, Nitrosurea, Etoposide (VP16), Tamoxifen,Raloxifene, Estrogen Receptor Binding Agents, Gemcitabien, Mavelbine,Farnesyl-protein transferase inhibitors, Transplatinum, 5-Fluorouracil,and Methotrexate, Temaxolomide (an aqueous form of DTIC), or any analogor derivative variant of the foregoing.

a. Cisplatinum

Agents that directly cross-link nucleic acids, specifically DNA, areenvisaged to facilitate DNA damage leading to a synergistic,anti-neoplastic combination with a mutant oncolytic virus. Cisplatinumagents such as cisplatin, and other DNA alkylating agents may be used.Cisplatinum has been widely used to treat cancer, with efficacious dosesused in clinical applications of 20 mg/m² for 5 days every three weeksfor a total of three courses. Cisplatin is not absorbed orally and musttherefore be delivered via injection intravenously, subcutaneously,intratumorally or intraperitoneally.

b. Daunorubicin

Daunorubicin hydrochloride, 5,12-Naphthacenedione,(8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10-methoxy-,hydrochloride; also termed cerubidine and available from Wyeth.Daunorubicin intercalates into DNA, blocked DNA-directed RNA polymeraseand inhibits DNA synthesis. It can prevent cell division in doses thatdo not interfere with nucleic acid synthesis.

In combination with other drugs it is included in the first-choicechemotherapy of acute myelocytic leukemia in adults (for induction ofremission), acute lymphocytic leukemia and the acute phase of chronicmyelocytic leukemia. Oral absorption is poor, and it must be givenintravenously. The half-life of distribution is 45 minutes and ofelimination, about 19 hr. the half-life of its active metabolite,daunorubicinol, is about 27 hr. daunorubicin is metabolized mostly inthe liver and also secreted into the bile (ca 40%). Dosage must bereduced in liver or renal insufficiencies.

Suitable doses are (base equivalent), intravenous adult, younger than 60yr. 45 mg/m²/day (30 mg/m2 for patients older than 60 yr.) for 1, 2 or 3days every 3 or 4 wk or 0.8 mg/kg/day for 3 to 6 days every 3 or 4 wk;no more than 550 mg/m² should be given in a lifetime, except only 450mg/m² if there has been chest irradiation; children, 25 mg/m² once aweek unless the age is less than 2 yr. or the body surface less than 0.5m, in which case the weight-based adult schedule is used. It isavailable in injectable dosage forms (base equivalent) 20 mg (as thebase equivalent to 21.4 mg of the hydrochloride). Exemplary doses may be10 mg/m², 20 Mg/m², 30 mg/m², 50 mg/m², 100 mg/m², 150 mg/m², 175 mg/m²,200 mg/m², 225 mg/m², 250 mg/m², 275 mg/m², 300 mg/m², 350 mg/m², 400mg/m², 425 mg/m², 450 mg/m², 475 mg/m², 500 mg/m². Of course, all ofthese dosages are exemplary, and any dosage in-between these points isalso expected to be of use in the invention.

9. Immunotherapy

In accordance with the invention, immunotherapy could be used incombination with a modulator of the JNK pathway in therapeuticapplications. Alternatively, immunotherapy could be used to modulateapoptosis via the JNK pathway. For example, anti-Gadd45β antibodies orantibodies to another component of the JNK pathway could be used todisrupt the function of the target molecule, thereby inhibiting Gadd45and increasing apoptosis. Alternatively, antibodies can be used totarget delivery of a modulator of the JNK pathway to a cell in needthereof. For example, the immune effector may be an antibody specificfor some marker on the surface of a tumor cell. Common tumor markersinclude carcinoembryonic antigen, prostate specific antigen, urinarytumor associate antigen, fetal antigen, tyrosinse (97), gp68, TAG-72,HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor, lamininreceptor, erb B and p155.

In an embodiment of the invention the antibody may be an anti-Gadd45βantibody. The antibody alone may serve as an effector of therapy or itmay recruit other cells to actually effect cell killing. The antibodyalso may be conjugated to a drug or toxin (chemotherapeutic,radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) andserve merely as a targeting agent. Alternatively, the effector may be alymphocyte carrying a surface molecule that interacts, either directlyor indirectly, with a target in a tumor cell, for example Gadd45β.Various effector cells include cytotoxic T cells and NK cells. Theseeffectors cause cell death and apoptosis. The apoptotic cancer cells arescavenged by reticuloendothelial cells including dendritic cells andmacrophages and presented to the immune system to generate anti-tumorimmunity (Rovere et al., 1999; Steinman et al., 1999). Immunestimulating molecules may be provided as immune therapy: for example,cytokines such as IL-2, IL-4, IL-12, GM-CSF, gamma-IFN, chemokines suchas MIP-1, MCP-1, IL-8 and growth factors such as FLT ligand. Combiningimmune stimulating molecules, either as proteins or using gene deliveryin combination with Gadd45 inhibitor will enhance anti-tumor effects.This may comprise: (i) Passive Immunotherapy which includes: injectionof antibodies alone; injection of antibodies coupled to toxins orchemotherapeutic agents; injection of antibodies coupled to radioactiveisotopes; injection of anti-idiotype antibodies; and finally, purging oftumor cells in bone marrow; and/or (ii) Active Immunotherapy wherein anantigenic peptide, polypeptide or protein, or an autologous or allogenictumor cell composition or “vaccine” is administered, generally with adistinct bacterial adjuvant (Ravindranath & Morton, 1991; Morton &Ravindranath, 1996; Morton et al., 1992; Mitchell et al., 1990; Mitchellet al, 1993) and/or (iii) Adoptive Immunotherapy wherein the patient'scirculating lymphocytes, or tumor infiltrated lymphocyltes, are isolatedin vitro, activated by lymphokines such as IL-2 or transduced with genesfor tumor necrosis, and readministered (Rosenberg et al., 1998; 1989).

10. Gene Therapy

Therapy in accordance with the invention may comprise gene therapy, inwhich one or more therapeutic polynucleotide is administered to apatient in need thereof. This can comprise administration of a nucleicacid that is a modulator of the JNK pathway, and may also compriseadministration of any other therapeutic nucleotide in combination with amodulator of the JNK pathway. One embodiment of cancer therapy inaccordance with the invention comprises administering a nucleic acidsequence that is an inhibitor of Gadd45β, such as a nucleic acidencoding a Gadd45β inhibitor polypeptide or an antisense Gadd45βsequence. Delivery of a vector encoding a JNK inhibitor polypeptide orcomprising an antisense JNK pathway modulator in conjunction with othertherapies, including gene therapy, will have a combinedanti-hyperproliferative effect on target tissues. A variety of proteinsare envisioned by the inventors as targets for gene therapy inconjunction with a modulator of the JNK pathway, some of which aredescribed below.

11. Clinical Protocol

A clinical protocol has been described herein to facilitate thetreatment of cancer using a modulator of the JNK pathway, such as aninhibitor of a Gadd45 protein, including the activity or expressionthereof by a Gadd45 gene. The protocol could similarly be used for otherconditions associated with a decrease in apoptosis. Alternatively, theprotocol could be used to assess treatments associated with increasedapoptosis by replacing the inhibitor of Gadd45 with an activator ofGadd45.

12. Therapeutic Kits

Therapeutic kits comprising a modulator of the JNK pathway are alsodescribed herein. Such kits will generally contain, in suitablecontainer means, a pharmaceutically acceptable formulation of at leastone modulator of the JNK pathway. The kits also may contain otherpharmaceutically acceptable formulations, such as those containingcomponents to target the modulator of the JNK pathway to distinctregions of a patient or cell type where treatment is needed, or any oneor more of a range of drugs which may work in concert with the modulatorof the JNK pathway, for example, chemotherapeutic agents.

The kits may have a single container means that contains the modulatorof the JNK pathway, with or without any additional components, or theymay have distinct container means for each desired agent. When thecomponents of the kit are provided in one or more liquid solutions, theliquid solution is an aqueous solution, with a sterile aqueous solutionbeing particularly preferred. However, the components of the kit may beprovided as dried powder(s). When reagents or components are provided asa dry powder, the powder can be reconstituted by the addition of asuitable solvent. It is envisioned that the solvent also may be providedin another container means. The container means of the kit willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which the monoterpene/triterpeneglycoside, and any other desired agent, may be placed and, preferably,suitably aliquoted. Where additional components are included, the kitwill also generally contain a second vial or other container into whichthese are placed, enabling the administration of separated designateddoses. The kits also may comprise a second/third container means forcontaining a sterile, pharmaceutically acceptable buffer or otherdiluent.

The kits also may contain a means by which to administer the modulatorsof the JNK pathway to an animal or patient, e.g., one or more needles orsyringes, or even an eye dropper, pipette, or other such like apparatus,from which the formulation may be injected into the animal or applied toa diseased area of the body. The kits of the present invention will alsotypically include a means for containing the vials, or such like, andother component, in close confinement for commercial sale, such as,e.g., injection or blow-molded plastic containers into which the desiredvials and other apparatus are placed and retained.

13. Gadd45 Compositions

Certain aspects of the current invention involve modulators of Gadd45.In one embodiment of the invention, the modulators may Gadd45 or othergenes or proteins. In particular embodiments of the invention, theinhibitor is an antisense construct. An antisense construct may comprisea full length coding sequence in antisense orientation and may alsocomprise one or more anti-sense oligonucleotides that may or may notcomprise a part of the coding sequence. Potential modulators of the JNKpathway, including modulators of Gadd45β, may include syntheticpeptides, which, for instance, could be fused to peptides derived fromthe Drosophila Antennapedia or HIV TAT proteins to allow free migrationthrough biological membranes; dominant negative acting mutant proteins,including constructs encoding these proteins; as well as natural andsynthetic chemical compounds and the like. Modulators in accordance withthe invention may also upregulate Gadd45, for example, by causing theoverexpression of a Gadd45 protein. Similarly, nucleic acids encodingGadd45 can be delivered to a target cell to increase Gadd45. The nucleicacid sequences encoding Gadd45 may be operably linked to a heterologouspromoter that may cause overexpression of the Gadd45.

Exemplary Gadd45 gene can be obtained from Genbank Accession No.NM-015675 for the human cDNA, NP 056490.1 for the human protein,NM-008655 for the mouse cDNA and NP-032681.1 for the mouse protein.Similarly, for Gadd45α nucleotide and protein sequences the GenbankAccession NOS. are: NM-001924 for the human cDNA; NP-001915 for thehuman protein; NM-007836 for the mouse cDNA and NP-031862.1 for themouse protein. For Gadd45γ nucleotide and protein sequences the GenbankAccession Nos. are: NM-006705 for the human cDNA, NP-006696.1 for thehuman protein, NM-011817 for the mouse cDNA and NP-035947.1 for themouse protein. Also forming part of the invention are contiguousstretches of nucleic acids, including about 25, about 50, about 75,about 100, about 150, about 200, about 300, about 400, about 55, about750, about 100, about 1250 and about 1500 or more contiguous nucleicacids of these sequences. The binding sites of the Gadd45 promotersequence, include the core binding sites of kB-1, kB-2 and kB-3, givenby any of these sequences may be used in the methods and compositionsdescribed herein.

Further specifically contemplated by the inventors are arrays comprisingany of the foregoing sequences bound to a solid support. Proteins ofGadd45 and other components of the JNK pathway may also be used toproduce arrays, including portions thereof comprising about 5, 10, 15,20, 25, 30, 40, 50, 60 or more contiguous amino acids of thesesequences.

14. Ribozymes

The use of ribozymes specific to a component in the JNK pathwayincluding Gadd45β specific ribozymes, is also a part of the invention.The following information is provided in order to complement the earliersection and to assist those of skill in the art in this endeavor.

Ribozymes are RNA-protein complexes that cleave nucleic acids in thesite-specific fashion. Ribozymes have specific catalytic domains thatpossess endonuclease activity (Kim and Cech, 1987; Gerlack et al., 1987;Forster and Symons, 1987). For example, a large number of ribozymesaccelerate phosphoester transfer reactions with a high degree ofspecificity, often cleaving only one of several phosphoesters in anoligonucleotide substrate (Cech et al., 1981; Michel and Westhof, 1990;Reinhold-Hurek and Shub, 1992). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

15. Proteins a. Encoded Proteins

Protein encoded by the respective gene can be expressed in any number ofdifferent recombinant DNA expression systems to generate large amountsof the polypeptide product, which can then be purified and used tovaccinate animals to generate antisera with which further studies may beconducted. In one embodiment of the invention, a nucleic acid thatinhibits a Gadd45 gene product or the expression thereof can be insertedinto an appropriate expression system. Such a nucleic acid may encode aninhibitor of Gadd45, including a dominant negative mutant protein, andmay also comprise an antisense Gad45 nucleic acid. The antisensesequence may comprise a full length coding sequence in antisenseorientation and may also comprise one or more anti-senseoligonucleotides that may or may not comprise a part of the codingsequence. Potential modulators of the JNK pathway, including modulatorsof Gadd45β, may include synthetic peptides, which, for instance, couldbe fused to peptides derived from a Drosophila Antennapedia or HIV TATproteins to allow free migration through biological membranes; dominantnegative acting mutant proteins, including constructs encoding theseproteins; as well as natural and synthetic chemical compounds and thelike.

Examples of other expression systems known to the skilled practitionerin the art include bacteria such as E. coli, yeast such as Pichiapastoris, baculovirus, and mammalian expression fragments of the geneencoding portions of polypeptide can be produced.

b. Mimetics

Another method for the preparation of the polypeptides according to theinvention is the use of peptide mimetics. Mimetics arepeptide-containing molecules which mimic elements of protein secondarystructure. See, for example, Johnson et al., “Peptide Turn Mimetics” inBIOTECHNOLOGY AND PHARMACY, Pezzuto et al., Eds., Chapman and Hall, NewYork (1993). The underlying rationale behind the use of peptide mimeticsis that the peptide backbone of proteins exists chiefly to orient aminoacid side chains in such a way as to facilitate molecular interactions,such as those of antibody and antigen. A peptide mimic is expected topermit molecular interactions similar to the natural molecule.

16. Pharmaceutical Formulations and Delivery

In an embodiment of the present invention, a method of treatment for acancer by the delivery of an expression construct comprising a Gadd45inhibitor nucleic acid is contemplated. A “Gadd45 inhibitor nucleicacid” may comprise a coding sequence of an inhibitor of Gadd45,including polypeptides, anti-sense oligonucleotides and dominantnegative mutants. Similarly, other types of inhibitors, includingnatural or synthetic chemical and other types of agents may beadministered. The pharmaceutical formulations may be used to treat anydisease associated with aberrant apoptosis levels.

An effective amount of the pharmaceutical composition, generally, isdefined as that amount of sufficient to detectably and repeatedly toameliorate, reduce, minimize or limit the extent of the disease or itssymptoms. More rigorous definitions may apply, including elimination,eradication or cure of the disease.

17. Methods of Discovering Modulators of the JNK Pathway

An aspect of the invention comprises methods of screening for any one ormore properties of Gadd45, including the inhibition of JNK or apoptosis.The modulators may act at either the protein level, for example, byinhibiting a polypeptide involved in the JNK pathway, or may act at thenucleic acid level by modulating the expression of such a polypeptide.Alternatively, such a modulator could affect the chemical modificationof a molecule in the JNK pathway, such as the phosphorylation of themolecule. The screening assays may be both for agents that modulate theJNK pathway to increase apoptosis as well as those that act to decreaseapoptosis. In screening assays for polypeptide activity, the candidatesubstance may first be screened for basic biochemical activity—e.g.,binding to a target molecule and then tested for its ability to regulateexpression, at the cellular, tissue or whole animal level. The assaysmay be used to detect levels of Gadd45 protein or mRNA or to detectlevels of protein or nucleic acids of another participant in the JNKpathway.

Exemplary procedures for such screening are set forth below. In all ofthe methods presented below, the agents to be tested could be either alibrary of small molecules (i.e., chemical compounds), peptides (e.g.,phage display), or other types of molecules.

a. Screening for Agents that Bind Gadd45β in vitro

96 well plates are coated with the agents to be tested according tostandard procedures. Unbound agent is washed away, prior to incubatingthe plates with recombinant Gadd45β proteins. After, additionalwashings, binding of Gadd45β to the plate is assessed by detection ofthe bound Gadd45, for example, using anti-Gadd45β antibodies andmethodologies routinely used for immunodetection (e.g. ELISA).

b. Screening for Agents that Inhibit Binding of Gadd45β to its MolecularTarget in the JNK Pathway

In certain embodiments, methods of screening and identifying an agentthat modulates the JNK pathway, are disclosed for example, that inhibitsor upregulates Gadd45β. Compounds that inhibit Gadd45 can effectivelyblock the inhibition of apoptosis, thus making cells more susceptible toapoptosis. This is typically achieved by obtaining the targetpolypeptide, such as a Gadd45 protein, and contacting the protein withcandidate agents followed by assays for any change in activity.

Candidate compounds can include fragments or parts ofnaturally-occurring compounds or may be only found as activecombinations of known compounds which are otherwise inactive. In apreferred embodiment, the candidate compounds are small molecules.Alternatively, it is proposed that compounds isolated from naturalsources, such as animals, bacteria, fungi, plant sources, includingleaves and bark, and marine samples may be assayed as candidates for thepresence of potentially useful pharmaceutical agents. It will beunderstood that the pharmaceutical agents to be screened could also bederived or synthesized from chemical compositions or man-made compounds.

Recombinant Gadd45β protein is coated onto 96 well plates and unboundprotein is removed by extensive washings. The agents to be tested arethen added to the plates along with recombinant Gadd45β-interactingprotein. Alternatively, agents are added either before or after theaddition of the second protein. After extensive washing, binding ofGadd45β to the Gadd45β-interacting protein is assessed, for example, byusing an antibody directed against the latter polypeptide andmethodologies routinely used for immunodetection (ELISA, etc.). In somecases, it might be preferable to coat plates with recombinantGadd45β-interacting protein and assess interaction with Gadd45β by usingan anti-Gadd45β antibody. The goal is to identify agents that disruptthe association between Gadd45β and its partner polypeptide.

c. Screening for Agents that Prevent the Ability of Gadd45β to BlockApoptosis

NF-κB-deficient cell lines expressing high levels of Gadd45β areprotected against TNFα-induced apoptosis. Cells (e.g., 3DO-IκBαM-Gadd45βclones) are grown in 96 well plates, exposed to the agents tested, andthen treated with TNFα Apoptosis is measured using standardmethodologies, for example, colorimetric MTS assays, PI staining, etc.Controls are treated with the agents in the absence of TNFα Inadditional controls, TNFα-sensitive NF-κB-null cells (e.g., 3DO-IκBαMcells), as well as TNFα-resistant NF-κB-competent cells (e.g., 3DO-Neo)are exposed to the agents to be tested in the presence or absence ofTNFα. The goal is to identify agents that induce apoptosis inTNFα-treated 3DO-IκBαM-Gadd45#, with animal toxicity in untreated cellsand no effect on TNFα-induced apoptosis in 3DO-IκBαM or 3DO-Neo cells.Agents that fit these criteria are likely to affect Gadd45β function,either directly or indirectly.

d. Screening for Agents that Prevent the Ability of Gadd45β to Block JNKActivation

Cell lines, treatments, and agents are as in c. However, rather than theapoptosis, JNK activation by TNFα is assessed. A potential complicationof this approach is that it might require much larger numbers of cellsand reagents. Thus, this type of screening might not be most useful as asecondary screen for agents isolated, for example, with other methods.

e. in vitro Assays

The present embodiment of this invention contemplates the use of amethod for screening and identifying an agent that modulates the JNKpathway. A quick, inexpensive and easy assay to run is a binding assay.Binding of a molecule to a target may, in and of itself, by inhibitory,due to steric, allosteric or charge-charge interactions. This can beperformed in solution or on a solid phase and can be utilized as a firstround screen to rapidly eliminate certain compounds before moving intomore sophisticated screening assays. The target may be either free insolution, fixed to a support, express in or on the surface of a cell.Examples of supports include nitrocellulose, a column or a gel. Eitherthe target or the compound may be labeled, thereby permittingdetermining of binding. In another embodiment, the assay may measure theenhancement of binding of a target to a natural or artificial substrateor binding partner. Usually, the target will be the labeled species,decreasing the chance that the labeling will interfere with the bindingmoiety's function. One may measure the amount of free label versus boundlabel to determine binding or inhibition of binding.

A technique for high throughput screening of compounds is described inWO 84/03564. In high throughput screening, large numbers of candidateinhibitory test compounds, which may be small molecules, naturalsubstrates and ligands, or may be fragments or structural or functionalmimetics thereof, are synthesized on a solid substrate, such as plasticpins or some other surface. Alternatively, purified target molecules canbe coated directly onto plates or supports for use in drug screeningtechniques. Also, fusion proteins containing a reactive region(preferably a terminal region) may be used to link an active region ofan enzyme to a solid phase, or support. The test compounds are reactedwith the target molecule, such as Gadd45β, and bound test compound isdetected by various methods (see, e.g., Coligan et al., CurrentProtocols in Immunology 1(2): Chapter 5, 1991).

Examples of small molecules that may be screened including small organicmolecules, peptides and peptide-like molecules, nucleic acids,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon-containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat modulate the JNK pathway. Further, in drug discovery, for example,proteins have been fused with antibody Fc portions for the purpose ofhigh-throughput screening assays to identify potential modulators of newpolypeptide targets. See, D. Bennett et al., Journal of MolecularRecognition, 8: 52-58 (1995) and K. Johanson et al., The Journal ofBiological Chemistry, 270, (16): 9459-9471 (1995).

In certain embodiments of the invention, assays comprise binding aGadd45 protein, coding sequence or promoter nucleic acid sequence to asupport, exposing the Gadd45β to a candidate inhibitory agent capable ofbinding the Gadd45β nucleic acid. The binding can be assayed by anystandard means in the art, such as using radioactivity, immunologicdetection, fluorescence, gel electrophoresis or colorimetry means. Stillfurther, assays may be carried out using whole cells for inhibitors ofGadd 45β through the identification of compounds capable of initiating aGadd45β-dependent blockade of apoptosis (see, e.g., Examples 8-11,below).

f. In Vivo Assays

Various transgenic animals, such as mice may be generated withconstructs that permit the use of modulators to regulate the signalingpathway that lead to apoptosis.

Treatment of these animals with test compounds will involve theadministration of the compound, in an appropriate form, to the animal.Administration will be, by any route that could be utilized for clinicalor non-clinical purposes including oral, nasal, buccal, or even topical.Alternatively, administration may be by intratracheal instillation,bronchial instillation, intradermal, subcutaneous, intramuscular,intraperitoneal or intravenous injection. Specifically contemplated aresystemic intravenous injection, regional administration via blood orlymph supply.

g. In Cyto Assays

The present invention also contemplates the screening of compounds fortheir ability to modulate the JNK pathway in cells. Various cell linescan be utilized for such screening assays, including cells specificallyengineered for this purpose. Depending on the assay, culture may berequired. The cell is examined using any of a number of different assaysfor screening for apoptosis or JNK activation in cells.

In particular embodiments of the present invention, screening maygenerally include the steps of:

-   -   (a) obtaining a candidate modulator of the JNK pathway, wherein        the candidate is potentially any agent capable of modulating a        component of the JNK pathway, including peptides, mutant        proteins, cDNAs, anti-sense oligonucleotides or constructs,        synthetic or natural chemical compounds, etc.;    -   (b) admixing the candidate agent with a cancer cell;    -   (c) determining the ability of the candidate substance to        modulate the JNK pathway, including either upregulation or        downregulation of the JNK pathway and assaying the levels up or        down regulation.

The levels up or down regulation will determine the extent to whichapoptosis is occurring in cells and the extent to which the cells are,for example, receptive to cancer therapy. In order to detect the levelsof modulation, immunodetection assays such as ELISA may be considered.

18. Methods of Assessing Modulators of Apoptotic Pathways InvolvingGadd45β In vitro and In Vivo

After suitable modulators of Gadd45β are identified, these agents may beused in accordance with the invention to increase or decrease Gadd45βactivity either in vitro and/or in vivo.

Upon identification of the molecular target(s) of Gadd45β in the JNKpathway, agents are tested for the capability of disrupting physicalinteraction between Gadd45β and the Gadd45β-interacting protein(s). Thiscan be assessed by employing methodologies commonly used in the art todetect protein-protein interactions, including immunoprecipitation, GSTpull-down, yeast or mammalian two-hybrid system, and the like. For thesestudies, proteins can be produced with various systems, including invitro transcription translation, bacterial or eukaryotic expressionsystems, and similar systems.

Candidate agents are also assessed for their ability to affect theGadd450-dependent inhibition of JNK or apoptosis. This can be tested byusing either cell lines that stably express Gadd45β (e.g.3DC-IκBαM-Gadd45β or cell lines transiently transfected with Gadd45βexpression constructs, such as HeLa, 293, and others. Cells are treatedwith the agents and the ability of Gadd45β to inhibit apoptosis or JNKactivation induced by various triggers (e.g., TNFα) tested by usingstandard methodologies. In parallel, control experiments are performedusing cell lines that do not express Gadd45β.

Transgenic mice expressing Gadd45β or mice injected with cell lines(e.g., cancer cells) expressing high levels of Gadd45β are used, eitherbecause they naturally express high levels of Gadd45β or because theyhave been engineered to do so (e.g., transfected cells). Animals arethen treated with the agents to be tested and apoptosis and/or JNKactivation induced by various triggers is analyzed using standardmethodologies. These studies will also allow an assessment of thepotential toxicity of these agents.

19. Methods of Treating Cancer with Modulators of Apoptotic PathwaysInvolving Gadd45β

This method provides a means for obtaining potentially any agent capableof inhibiting Gadd45β either by way of interference with the function ofGadd45β protein, or with the expression of the protein in cells.Inhibitors may include: naturally-occurring or synthetic chemicalcompounds, particularly those isolated as described herein, anti-senseconstructs or oligonucleotides, Gadd45β mutant proteins (i.e., dominantnegative mutants), mutant or wild type forms of proteins that interferewith Gadd45β expression or function, anti-Gadd45β antibodies, cDNAs thatencode any of the above mentioned proteins, ribozymes, syntheticpeptides and the like.

a. in vitro Methods

i) Cancer cells expressing high levels of Gadd45β, such as variousbreast cancer cell lines, are treated with candidate agent and apoptosisis measured by conventional methods (e.g., MTS assays, PI staining,caspase activation, etc.). The goal is to determine whether theinhibition of constitutive Gadd45β expression or function by theseagents is able to induce apoptosis in cancer cells. ii) In separatestudies, concomitantly with the agents to be tested, cells are treatedwith TNFα or the ligands of other “death receptors” (DR) (e.g., Fasligand binding to Fas, or TRAIL binding to both TRAIL-R1 and -R2). Thegoal of these studies is to assess whether the inhibition of Gadd45βrenders cancer cells more susceptible to DR-induced apoptosis. iii) Inother studies, cancer cells are treated with agents that inhibit Gadd45βexpression or function in combination with conventional chemotherapyagents or radiation. DNA damaging agents are important candidates forthese studies. However, any chemotherapeutic agent could be used. Thegoal is to determine whether the inhibition of Gadd45β renders cancercells more susceptible to apoptosis induced by chemotherapy orradiation.

b. In Vivo Methods

The methods described above are used in animal models. The agents to betested are used, for instance, in transgenic mice expressing Gadd45β ormice injected with tumor cells expressing high levels of Gadd45β, eitherbecause they naturally express high levels of Gadd45β or because theyhave been engineered to do so (e.g., transfected cells). Of particularinterest for these studies, are cell lines that can form tumors in mice.The effects of Gadd45β inhibitors are assessed, either alone or inconjunction with ligands of DRs (e.g. TNFα and TRAIL), chemotherapyagents, or radiation on tumor viability. These assays also allowdetermination of potential toxicity of a particular means of Gadd45βinhibition or combinatorial therapy in the animal.

20. Regulation of the gadd45β Promoter by NF-κB

κB binding sites were identified in the gadd45β promoter. The presenceof functional κB sites in the gadd45β promoter indicates a directparticipation of NF-κB complexes in the regulation of Gadd45β, therebyproviding an important protective mechanism by NF-κB.

21. Isolation and Analysis of the gadd45β Promoter

A BAC clone containing the murine gadd45β gene was isolated from a 129SB mouse genomic library (mouse ES I library; Research Genetics),digested with Xho I, and ligated into the XhoI site of pBluescript IISK- (pBS; Stratagene). A pBS plasmid harboring the 7384 bp Xho Ifragment of gadd45β (pBS-014D) was subsequently isolated and completelysequenced by automated sequencing at the University of Chicagosequencing facility. The TRANSFAC database (Heinemeyer et al., 1999) wasused to identify putative transcription factor-binding DNA elements,whereas the BLAST engine (Tatusova et al., 1999) was used for thecomparative analysis with the human promoter.

22. Plasmids

The pMT2T, pMT2T-p50, and pMT2T-RelA expression plasmids were describedpreviously (Franzoso et al., 1992). To generate the gadd45β-CAT reporterconstructs, portions of the gadd45β promoter were amplified frompBS-014D by polymerase chain reaction (PCR) using the following primers:5′-GGATAACGCGTCACCGTCCTCAAACTTACCAAACGTTTA-3′(SEQ ID NO: 16) and5′-GGATGGATATCCGAAATTAATCCAAGAAGACAGAGATGAAC-3′ (SEQ ID NO: 17)(−592/+23-gadd45β, MluI and EcoRV sites incorporated into sense andanti-sense primers, respectively, are underlined);5′-GGATAACGCGTTAGAGCTCTCTGGCTTTTCTAGCTGTC-3′ (SEQ ID NO: 18)and5′GGATGGATATCCGAAATTAATCCAAGAAGACAGAGATGAAC-3′ (SEQ ID NO: 19)(−265/+23-gadd45β); 5′-GGATAACGCGTAAAGCGCATGCCTCCAGTGGCCACG-3′ (SEQ IDNO: 20) and 5′-GGATGGATATCCGAAATTAATCCAAGAAGACAGAGATGAAC-3′ (SEQ ID NO:21) (−103/+23-gadd45γ); 5′-GGATAACGCGTCACCGTCCTCAAACTTACCAAACGTTTA-3′(SEQ ID NO: 22) and 5′-GGATGGATATCCAAGAGGCAAAAAAACCTTCCCGTGCGA-3′ (SEQID NO: 23) (−592/+139-gadd45γ);5′-GGATAACGCGTTAGAGCTCTCTGGCTTTTCTAGCTGTC-3′ (SEQ ID NO: 24) and5′-GGATGGATATCCAAGAGGCAAAAAAACCTTCCCGTGCGA-3′ (SEQ ID NO: 25)(−265/+139-gadd45γ). PCR products were digested with MIul and EcoRV andligated into the MluI and SmaI sites of the promoterless pCAT3-Basicvector (Promega) to drive ligated into the MluI and SmaI sites of thepromoterless pCAT2-Basic vector (Promega) to drive expression of thechloramphenicol acetyl-transferase (CAT) gene. All inserts wereconfirmed by sequencing. To generate −5407/−23-gadd45β-CAT and−3465/+23-gadd45β-CAT, pBS-014D was digested with XhoI or EcoNI,respectively, subjected to Klenow filling, and further digested withBssHII. The resulting 5039 bp XhoI-BssHII and 3097 bp EcoNI-BssH IIfragments were then independently inserted between a filled-in MluI siteand the BssHII site of −592/+23-gadd45β-CAT. The two latter constructscontained the gadd45β promoter fragment spanning from either −5407 or−3465 to −368 directly joined to the −38/+23 fragment. Both reporterplasmids contained intact κB-1, κB-2, and κB-3 sites (see FIG. 10).

κB-1M-gadd45β-CAT, κB-2M-gadd45β-CAT, and κB-3M-gadd45β-CAT wereobtained by site-directed mutagenesis of the −592+23-gadd45(3-CATplasmid using the QuikChange™ kit (Stratagene) according to themanufacturer's instructions. The following base substitution wereintroduced: 5′-TAGGGACTCTCC-2′ (SEQ ID NO: 26) to 5′-AATATTCTCTCC-3′(SEQ ID NO: 27) (κB-1M-gadd45β-CAT; κB sites and their mutatedcounterparts are underlined; mutated nucleotides are in bold);5′-GGGGATTCCA-3′ (SEQ ID NO: 28) to 5′-ATCGATTCCA-3′ (SEQ ID NO: 29)(κB-2M-gadd45β-CAT); and 5′-GGAAACCCCG-3′ (SEQ ID NO: 30) to5′-GGAAATATTG-3′ (SEQ ID NO: 31) (κB-3M-gadd45β-CAT).κB-1/2-gadd45β-CAT, containing mutated κB-1 and κB-2 sites, was derivedfrom κB-2M-gadd45β-CAT by site-directed mutagenesis of κB-1, asdescribed above. With all constructs, the −592/+23 promoter fragment,including mutated κB elements, and the pCAT-3-Basic region spanning fromthe SmaI cloning site to the end of the CAT poly-adenylation signal wereconfirmed by sequencing.

Δ56-κB-1/2-CAT, Δ56-κB-3-CAT, and Δ56-κB-M-CAT reporter plasmids wereconstructed by inserting wild-type or mutated oligonucleotides derivedfrom the mouse gadd45β promoter into Δ56-CAT between the BglII and XhoIsites, located immediately upstream of a minimal mouse c-fos promoter.The oligonucleotides used were:5′-GATCTCTAGGGACTCTCCGGGGACAGCGAGGGGATTCCAGACC-3′ (SEQ ID NO: 32)(κB-1/2-CAT; κB-1 and κB-2 sites are underlined, respectively);5′-GATCTGAATTCGCTGGAAACCCCGCAC-3′ (SEQ ID NO: 33) (κB-3-CAT; κB-3 isunderlined); and 5′-GATCTGAATTCTACTTACTCTCAAGAC-3′ (SEQ ID NO: 34)(κB-M-CAT).

23. Transfections, CAT Assays, and Electrophoretic Mobility Shift Assays(EMSAs)

Calcium phosphate-mediate transient transfection of NTera-2 cells andCAT assays, involving scintillation vial counting, were performed asreported previously (Franzoso et al., 1992, 1993). EMSA, supershiftinganalysis, and antibodies directed against N-terminal peptides of humanp50 and RelA were as described previously (Franzoso et al., 1992). Wholecell extracts from transfected NTera-2 cells were prepared by repeatedfreeze-thawing in buffer C (20 mM HEPES [pH 7.9], 0.2 MM EDTA; 0.5 mMMgCl₂, 0.5 M NaCl, 25% glycerol, and a cocktail of protease inhibitors[Boehringer Mannheim]), followed by ultracentrifugation, as previouslydescribed.

24. Generation and Treatments of BJAB Clones and Oropidium IodideStaining Assays

To generate stable clones, BJAB cells were transfected withpcDNA-HA-Gadd45β or empty pcDNA-HA plamids (Invitrogen), and 24 hourslater, subjected to selection in G418 (Cellgro; 4 mg/ml). Resistantclones where expanded and HA-Gadd45β expression was assessed by Westernblotting using anti-HA antibodies or, to control for loading,anti-β-actin antibodies.

Clones expressing high levels of HA-Gadd45β and control HA clones (alsoreferred to as Neo clones) were then seeded in 12-well plates and leftuntreated or treated with the agonistic anti-Fas antibody APO-1 (1μg/ml; Alexis) or recombinant TRAIL (100 ng/ml; Alexis). At the timesindicated, cells were harvested, washed twice in PBS and incubatedovernight at 4° C. in a solution containing 0.1% Na citrate (pH 7.4), 50μg/ml propidium iodide (PI; Sigma), and 0.1% Triton X-100. Cells werethen examined by flow cytometry (FCM) in both the FL-2 and FL-3channels, and cells with DNA content lesser than 2N (sub-G1 fraction)were scored as apoptotic.

For the protective treatment with the JNK blocker SP600125 (Calbiochem),BJAB cells were left untreated or pretreated for 30 minutes with variousconcentrations of the blocker, as indicated, and then incubated for anadditional 16 hours with the agonistic anti-Fas antibody APO-1 (1μg/ml). Apoptosis was scored in PI assays as described herein.

25. Treatments, Viral Tranduction, and JNK Kinase Assays with JNK NullFibroblasts

JNK null fibroblast—containing the simultaneous deletion of the jnk1 andjnk2 genes—along with appropriate control fibroblasts, were obtainedfrom Dr. Roger Davis (University of Massachusetts). For cytotoxicityexperiments, knockout and wild-type cells were seeded at a density of10,000 cells/well in 48-well plates, and 24 hours later, treated withTNFα alone (1,000 U/ml) or together with increasing concentrations ofcycloheximide (CHX). Apoptosis was monitored after a 8-hour treatment byusing the cell death detection ELISA kit (Boehringer-Roche) according tothe manufacturer's instructions. Briefly, after lysing the cellsdirectly in the wells, free nucleosomes in cell lysates were quantifiedby ELISA using a biotinylated anti-histone antibody. Experiments werecarried out in triplicate.

The MIGR1 retroviral vector was obtained from Dr. Harinder Singh(University of Chicago). MIGR1-JNKK2-JNK1, expressing constitutivelyactive JNK1, was generated by excising the HindIII-BglII fragment ofJNKK2-JNK1 from pSRα-JNKK2-JNK1 (obtained from Dr. Anning Lin,University of Chicago), and after filling-in this fragment by Klenow'sreaction, inserting it into the filled-in XhoI site of MIGR1. High-titerretroviral preparations were obtained from Phoenix cells that had beentransfected with MIGR1 or MIGR1-JNKK2-JNK1. For viral transduction,mutant fibroblasts were seeded at 100,000/well in 6-well plates andincubated overnight with 4 ml viral preparation and 1 ml complete DMEMmedium in 5 μg/ml polybrene. Cells were then washed with completemedium, and 48 hours later, used for cytotoxic assays.

For JNK kinase assays, cells were left untreated or treated with TNFα(1,000 U/ml) for 10 minutes, and lysates were prepared in a buffercontaining 20 mM HEPES (pH 8.0), 350 mM NaCl, 20% glycerol, 1% NP-40, 1mM MgCl₂, 0.2 mM EGTA, 1 mM DTT, 1 mM Na₃VO₄, 50 mM NaF, and proteaseinhibitors. JNK was immunoprecipitated from cell lysates by using acommercial anti-JNK antibody (BD Pharmingen) and kinase assays wereperformed as described for FIGS. 6 and 7 using GST-c-Jun substrates.

26. Treatment of WEHI-231 Cells and Electrophoretic Mobility ShiftAssays

WEHI-231 cells were cultured in 10% FBS-supplemented RPMI mediumaccording to the recommendations of the American Type Culture Collection(ATCC). For electrophoretic mobility shift assays (EMSAs), cells weretreated with 40 μg/ml lypopolysaccharide (LPS; Escherichia coli serotype0111:B4), and harvested at the times indicated. Cell lysates wereprepared by repeated freeze-thawing in buffer C (20 mM HEPES [pH 7.9],0.2 mM EDTA, 0.5 mM DTT, 1.5 mM MgCl₂, 0.42 M NaCl, 25% glycerol, andprotease inhibitors) followed by ultracentrifugation. For in vitro DNAbinding assays, 2 μl cell extracts were incubated for 20 minutes withradiolabeled probes derived from each of the three κB sites found in themurine gadd45β promoter. Incubations were carried out in buffer D (20 mMHEPES [pH 7.9], 20% glycerol, 100 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5mM PMSF) containing 1 μg/ml polydI-dC and 0.1 μg/ml BSA, and DNA-bindingcomplexes were resolved by polyacrilamide gel electrophoresis. Forsupershifts, extracts were pre-incubated for 10 minutes with 1 μl ofantibodies reacting with individual NF-κB subunits.

27. Treatments of BT-20 and MDA-MD-231 cells

Breast cancer cell lines were cultured in complete DMEM mediumsupplemented with 10% FCS and seeded at 100,000/well in 12-well plates.After 24 hours, cultures were left untreated or pre-treated for 1 hourwith the indicated concentrations of the SP600125 inhibitor(Calbiochem), after which the NF-κB inhibitors prostaglandin A1, CAPE,or parthenolide (Biomol) were added as shown in FIG. 20. At theindicated times, cell death was scored morphologically by lightmicroscopy.

28. Co-immunoprecipitations with 293 Cell Lysates

293 cells were transfected by the calcium phosphate method with 15 μgpcDNA-HA plasmids expressing either full-length (FL) human MEKK1, MEKK3,GCK, GCKR, ASK1, MKK7/JNKK2, and JNK3, or murine MEKK4 and MKK4/JNKK1along with 15 μg pcDNA-FLAG-Gadd45β—expressing FL murine Gadd45β—orempty pcDNA-FLAG vectors. pcDNA vectors (Invitrogen). 24 hours aftertransfection, cells were harvested, and cell lysates were prepared byresuspending cell pellets in CO-IP buffer (40 mM TRIS [pH 7.4], 150 mMNaCl, 1% NP-40, 5 mM EGTA, 20 mM NaF, 1 mM Na₃VO₄, and proteaseinhibitors) and subjecting them to ultracentrifugation.

For co-immunoprecipitations (co-IP), 200 μg cell lysate were incubatedwith anti-FLAG(M2)-coated beads (Sigma) in CO-IP buffer for 4 hours at4° C. After incubation, beads were washed 4 times and loaded ontoSDS-polyacrylamide gels, and Western bots were performed by usinganti-HA antibodies (Santa Cruz).

29. GST Fusion Proteins Constructions and GST Pull-Down Assays

Murine Gadd45β and human JNKK2 were cloned into the EcoRi and BamHIsites of the pGEX-3× and pGEX-2T bacterial expression vectors (both fromAmersham), respectively. These constructs and the pGEX-3X vector anwithout insert were introduced into E. coli BL21 cells in order toexpress GST-Gadd45β, GST-JNKK2, and GST proteins. Following inductionwith 1 mM IPTG, cells were lysed by sonication in PBS and thenprecipitated with glutathione-sepharose beads (Sigma) in the presence of1% Triton X-100, and washed 4 times in the same buffer.

In vitro transcription and translation reactions were carried out byusing the TNT coupled reticulocyte lysate system (Promega) according tothe manufacturer's instructions in the presence of [³⁵S]methionine. Toprime in vitro reactions, cDNAs were cloned into the pBluescript (pBS)SK− plasmid (Stratagene). FL murine MEKK4 was cloned into the SpeI andEcoRI sites of pBS and was transcribed with the T3 polymerase; FL humanJNKK2, FL murine JNKK1, and FL human ASK1, were cloned into theXbaI-EcoRI, NotI-EcoRI, and XbaI-ApaI sites of pBS, respectively, andwere transcribed by using the T7 polymerase. pBS-C-ASK1—encoding aminoacids 648-1375 of human ASK1—was derived from pBS-FL-ASK1 by excision ofthe EarI and XbaI fragment of ASK1 and insertion of the followingoligonucleotide linker: 5′-CGCCACCATGGAGATGGTGAACACCAT-3′ (SEQ ID NO:47). N-ASK1—encoding the 1-756 amino acid fragment of ASK1—was obtainedby priming the in vitro transcription/translation reaction withpBS-FL-ASK1 digested with PpuMI.

pBS plasmids expressing N-terminal deletions of human JNKK2 weregenerated by digestion of pBS-FL-JNKK2 with BamHI and appropriaterestriction enzymes cleaving within the coding sequence of JNKK2 andreplacement of the excised fragments with an oligonucleotide containing(5′ to 3′): a BamHI site, a Kozak sequence, an initiator ATG, and anucleotide sequence encoding between 7 and 13 residues of JNKK2.resulting pBS plasmids encoded the carboxy-terminal amino acidic portionof that is indicated in FIG. 28. To generate JNKK2 C-terminal deletions,pBS-FL-JNKK2 was linearized with SacII, PpuMI, NotI, XcmI, BsgI, BspEI,BspHI, or PflMI, prior to be used to prime in vitrotranscription/translation reactions. The resulting polypeptide productscontain the amino-terminal amino acidic sequence of JNKK2 that isindicated in FIG. 28.

To generate Gadd45β polypeptides, in vitro reactions were primed withpBS-GFP-Gadd450 plasmids, encoding green fluorescent protein (GFP)directly fused to FL or truncated Gadd45β. To obtain these plasmids,pBS-Gadd45β(FL), pBS-Gadd45β (41-160), pBS-Gadd45 (60-160), pBS-Gadd45β(69-160), pBS-Gadd45β (87-160), and pBS-Gadd45β (113-160)—encoding thecorresponding amino acid residues of murine Gadd45β were generated—bycloning appropriate gadd45β cDNA fragments into the XhoI and HindIIIsites of pBS SK−. These plasmids, encoding either FL or truncatedGadd45β, were then opened with KpnI and XhoI, and the excised DNAfragments were replaced with the KpnI-BsrGI fragment of pEGFP-N1(Clontech; containing the GFP-coding sequence) directly joined to thefollowing oligonucleotide linker:5′-GTACAAGGGTATGGCTATGTCAATGGGAGGTAG-3′ (SEQ ID NO: 48). Theseconstructs were designated as pBS-GFP-Gadd45β. Gadd45β C-terminaldeletions were obtained as described for the JNKK2 deletions by usingpBS-GFP-Gadd45β(FL) that had been digested with the NgoMI, SphI, orEcoRV restriction enzymes to direct protein synthesis in vitro. Theseplasmids encoded the 1-134, 1-95, and 1-68 amino acid fragments ofGadd45β, respectively. All pBS-Gadd45β constructs were transcribed usingthe T7 polymerase.

For GST pull-down experiments, 5 μl of in vitro-translated andradio-labeled proteins were mixed with glutathione beads carrying GST,GST-JNKK2 (only with Gadd45β translation products), or GST-Gadd45β (onlywith ASK1, MEKK4, JNKK1, and JNKK2 translation products) and incubatedfor 1 hour at room temperature in a buffer containing 20 mM TRIS, 150 mMNaC, and 0.2% Triton X-100. The beads were then precipitated and washed4 times with the same buffer, and the material was separated by SDSpolyacrylamide gel electrophoresis. Alongside of each pair of GST andGST-JNKK2 or GST-Gadd45β beads were loaded 2 μl of crude in vitrotranscription/translation reaction (input).

30. Kinase Assays

To test the inhibitory effects of recombinant Gadd45β proteins on kinaseactivity, HEK-293 cells were transfected by using the calcium phosphatemethod with 1 to 10 □g of pcDNA-FLAG-JNKK2, pcDNA-FLAG-JNKK1,pcDNA-FLAG-MKK3b or pcDNA-FLAG-ASK1, and empty pcDNA-FLAG to 30 □g totalDNA. 24 hours later, cells were treated for 20 minutes with human TNFα(1,000 U/ml) or left untreated, harvested, and then lysed in a buffercontaining 20 mM HEPES (pH 8.0), 350 mM NaCl, 20% glycerol, 1% NP-40, 1mM MgCl₂, 0.2 mM EGTA, 1 mM DTT, 1 mM Na₃VO₄, 50 mM NaF, and proteaseinhibitors, and subjected to ultracentrifugation. Immunoprecipitationswere performed using anti-FLAG(M2)-coated beads (Sigma) and 200 □g celllysates. After immunoprecipitation, beads were washed twice in lysisbuffer and twice more in kinase buffer. To assay for kinase activity ofimmunoprecipitates, beads were pre-incubated for 10 minutes withincreasing amounts of recombinant His₆-Gadd45β (His₆ disclosed as SEQ IDNO: 46), GST-Gadd45β, or control proteins in 30 □l kinase buffercontaining 10 M ATP and 10 μCi [³²P]⁻□ATP, and then incubated for 1additional hour at 30° C. with 1 □g of the appropriate kinase substrate,as indicated. the following kinase buffers were used: 20 mM HEPES, 20 mMMgCl₂, 20 mM β-glycero-phosphate, 1 mM DTT, and 50 βM Na₃VO₄ for JNKK2;20 mM HEPES, 10 mM MgCl₂, 20 mM β-glycero-phosphate, and 0.5 mM DTT forJNKK1; 25 mM HEPES, 25 mM MgCl₂, 25 mM P-glycero-phosphate, 0.5 mM DTT,and 50 μM Na₃VO₄ for MKK3; 20 mM Tris HCl, 20 mM MgCl₂, 20 mMβ-glycero-phosphate, 1 mM DTT, and 50 μM Na₃VO₄ for ASK1.

To assay activity of endogenous kinases, immunoprecipitations wereperformed by using appropriate commercial antibodies (Santa Cruz)specific for each enzyme and cell lysates obtained from3DO-IκBαM-Gadd45β and 3DO-IκBαM-Hygro clones prior and after stimulationwith TNFα (1,000 U/ml), as indicated. Kinase assays were performed asdescribed above, but without pre-incubating immunoprecipitates withrecombinant Gadd45β proteins.

31. Cytoprotection Assays in RelA Knockout Cells and pEGFP-Gadd45βConstructs

Plasmids expressing N- and C-terminal truncations of murine Gadd45β wereobtained by cloning appropriate gadd45β cDNA fragments into the XhoI andBamHI sites of pEGFP-N1 (Clontech). These constructs expressed theindicated amino acids of Gadd45β directly fused to the N-terminus ofGFP. For cytoprotection assays, GFP-Gadd45β-coding plasmids or emptypEGFP were transfected into RelA−/− cells by using Superfect (Qiagen)according to the manufacturer's instructions, and 24 hours later,cultures were treated with CHX alone (0.1 μg/ml) or CHX plus TNFα (1,000U/ml). After a 12-hour treatment, live cells adhering to tissue cultureplates were counted and examined by FCM to assess GFP positivity.Percent survival values were calculated by extrapolating the totalnumber of live GFP⁺ cells present in the cultures that had been treatedwith CHX plus TNFα relative to those treated with CHX alone.

32. Plasmids in Example 12.

-   -   pcDNA-HA-GCKR, pCEP-HA-MEKK1, pcDNA-HA-ASK1, pCMV5-HA-MEKK3,        pCMV5-HA-MEKK4, pcDNA-HA-MEK1, pMT3-HA-MKK4, pSRα-HA-JNK1,        pMT2T-HA-JNK3, pcDNA-HA-ERK1, pSRα-HA-ERK2, pcDNA-FLAG-p38□,        pcDNA-FLAG-p38□, pcDNA-FLAG-p38γ, and pcDNA-FLAG-p38δ were        provided by A. Leonardi, H. Ichijo, J. Landry, R.        Vaillancourt, P. Vito, T. H. Wang, J. Wimalasena, and H. Gram.        pcDNA-HA-Gadd45β, pGEX-JNK1, pET28-His₆/T7-JIP1 (expressing the        MKK7-binding domain of JIP1b), and pProEx-1.His_(□)-EF3        (expressing edema factor 3). All other FLAG- or HA-coding        constructs were generated using pcDNA (Invitrogen). For        bacterial expression, sub-clonings were in the following        vectors: His₆/T7-Gadd45β (His disclosed as SEQ NO: 46) in pET-28        (Novagen); His₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46) in        pProEx-1.H₆ ²⁰; GST-p38α, GST-MKK7, and GST-Gadd45β in pGEX        (Amersham). To prime in vitro transcription/translations,        pBluescript(BS)-MEKK4, pBS-ASK1, and pBS-MKK7 were generated        (FIG. 26); pBS-based plasmids expressing N-terminal truncations        and polypeptidic fragments of human MKK7. To enhance        radio-labeling, the latter peptides were expressed fused to        enhanced green fluorescent protein (eGFP, Clontech). ASK1¹⁻⁷⁵⁷        (encoding amino acids 1-757 of ASK1) and C-terminal MKK7        truncations were obtained by linearizing pBS-ASK1 and pBS-MKK7,        respectively, with appropriate restriction enzymes.

33. Treatments and Apoptosis Assays

Treatments were as follows: murine TNFα (Peprotech), 1,000 U/ml (FIG.27) or 10 U/ml (FIG. 30); human TNFα (Peprotech), 2,000 U/ml; PMA plusionomycin (Sigma), 100 ng/ml and 1 μM, respectively. In FIG. 30,pre-treatment with HIV-TAT peptides (5 μM) or DMSO was for 30 minutesand incubation with TNFα was for an additional 7 and 3.5 hours,respectively. Apoptosis was measured by using the Cell Death DetectionELISA^(PLUS) kit (Roche).

34. Binding Assays, Protein Purification, and Kinase Assays

GST precipitations with in vitro-translated proteins or purifiedproteins (FIG. 26-30), and kinase assays were performed. His₆/T7-Gadd45β(His₆ disclosed as SEQ ID NO: 46), His₆/T7-JIP1 (His₆disclosed as SEQ IDNO: 46), His₆-Gadd45β (His₆ disclosed as SEQ ID NO: 46), His₆-EF3(His₆disclosed as SEQ ID NO: 46), and GST proteins were purified frombacterial lysates as detailed elsewhere, and dialyzed against buffer A¹⁹(FIG. 28) or 5 mM Na⁺ phosphate buffer (pH 7.6; FIG. 28, 30). Kinasepre-incubation with recombinant proteins was for 10 minutes (FIG. 28,30), and GST-Gadd45β pre-incubation with peptides or DMSO (−) was for anadditional 20 minutes (FIG. 30). MKK7 phosphorylation was monitored byperforming immunoprecipitations with anti-P-MKK7 antibodies (developedat Cell Signaling) followed by Western blots with anti-total MKK7antibodies. For co-immunoprecipitations, extracts were prepared in IPbuffer.

35. Antibodies

The anti-MKK7 antibodies were: FIG. 27, kinase assays (goat; SantaCruz); FIG. 27, Western blots, and FIG. 3 a, top right,immunoprecipitations (rabbit; Santa Cruz); FIG. 28, top left, Westernblot (mouse monoclonal; BD Pharmingen). Other antibodies were: anti-FLAGfrom Sigma; anti-P-MKK4, anti-P-MKK3/6, anti-P-MEK1/2, anti-total MKK3,and anti-total MEK1/2 from Cell Signaling; anti-T7 from Novagen;anti-HA, anti-total MKK4, anti-total ASK1 (kinase assays and Westernblots), and anti-total MEKK1 (kinase assays, Western blots, andco-immunoprecipitations) from Santa Cruz. There was an anti-Gadd45βmonoclonal antibody (5D2.2).

36. Peptide Intracellular Incorporation Assays, Treatments, andApoptosis Assays

Treatments were as follows: murine TNFα (Peprotech), 1,000 U/ml, 10U/ml, or 1,000 U/ml plus 0.3 μg/ml cycloheximide (CHX; FIG. 33); humanTNFα (Peprotech), 2,000 U/ml; PMA plus ionomycin (Sigma), 100 ng/ml and1 μM, respectively. Treatments with H₂O₂ and sorbitol were as describedpreviously. In FIG. 33, pre-treatment with HIV-TAT peptides (5 μM) orDMSO was for 30 minutes and incubation with TNFα was for an additional 4and 3.5 hours, respectively. In FIG. 33, peptides were used at 10 μM andincubation with TNFα was for 4 hours. Apoptosis was measured by usingthe Cell Death Detection ELISA PLUS kit (Roche). To assess intracellularincorporation, peptides were labeled with FITC either at the N-terminusduring synthesis or after HPLC purification by using the FluoReporterFITC protein labeling kit (Molecular Probes). Cells were then incubatedwith 5 μM peptides for 20 minutes, subjected to trypsinization, washedthree times with PBS, and examined by FCM or confocal microscopy.

37. Generation of gadd45β^(−/−) Fibroblasts

Gadd45β null mice were generated with the help of the Transgenic andKnockout facility at the University of Chicago by using standardhomologous recombination-based technology in ES cells. MEFs wereisolated from mouse embryos at day 14 post-coitum.

38. Methods to Identify Peptide 2-Interacting Factors

Methods to identify peptide 2-interacting factors include techniquessuch as two-hybrid system, phage display, affinity purification, andGST-pull downs.

Phage display describes a selection technique in which a peptide orprotein is expressed as a fusion with a coat protein of a bacteriophage,resulting in display of the fused protein on the exterior surface of thephage virion, while the DNA encoding the fusion resides within thevirion. Phage display has been used to create a physical linkage betweena vast library of random peptide sequences to the DNA encoding eachsequence, allowing rapid identification of peptide ligands for a varietyof target molecules (antibodies, enzymes, cell-surface receptors, signaltransducers and the like) by an in vitro selection process called“panning”. Commercially available systems such as Ph.D.™ Phage DisplayPeptide Library Kits (New England Biolabs, MA) can be used.

Affnity column-based purification systems can also be used to identifyinteracting proteins. Commercially available affinity purificationsystems such as the Strep-tag™ purification system based on the highlyselective binding of engineered streptavidin, called Strep-Tactin, toStrep-tag II fusion proteins are useful (IBA GmbH, Germany). Thistechnology allows one-step purification of recombinant protein underphysiological conditions, thus preserving its bioactivity. The Strep-tagsystem can be used to purify functional Strep-tag II proteins from anyexpression system including baculovirus, mammalian cells, yeast, andbacteria. Unique Strep-Tactin affinity columns have been developed forthis purpose and the corresponding operating protocols are describedbelow. Because of its small size, Strep-tag generally does not interferewith the bioactivity of the fusion partner.

The yeast two-hybrid system is a widespread method used to studyprotein-protein interactions. In this system, one protein, the “bait”molecule, is fused to a DNA-binding domain (e.g., Escherichia coli LexAprotein), and the other partner, the “prey” molecule, is fused to anactivation domain (e.g., yeast GAL4 protein). When these two hybridproteins interact, a bipartite transcription factor is reconstituted andcan transactivate reporter genes, such as lacZ (encodingbeta-galactosidase) or his3 (encoding imidazole acetol phosphatetransaminase enzyme), which are downstream of DNA-binding sites for thebait protein's DNA-binding domain. The system is also of great use fordetecting and characterizing new binding partners for a specific proteinthat is fused to the DNA-binding domain. This is achieved by screening alibrary of cDNAs fused to the sequence of the activation domain. In atypical screening protocol, the plasmid DNA from each yeast clone mustbe isolated in order to identify the cDNA. Commercially availablesystems such as Checkmate™ Mammalian Two-Hybrid System (Promega,Madison, Wis.) can be used to identify interacting factors.

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1. A polypeptide molecule comprising a Gadd45β binding region of JNKK2consisting of the amino acid sequence from positions 132-156(GPVWKMRFRKTGHVIAVKQMRRSGN) (SEQ ID NO: 4) and further comprising aheterologous peptide.
 2. The polypeptide of claim 1, wherein theheterologous peptide renders the polypeptide cell permeable.
 3. Thepolypeptide of claim 1, wherein the polypeptide is a fusion polypeptide.4. The polypeptide of claim 1 wherein the heterologous peptide comprisesa TAT peptide.
 5. The polypeptide of claim 1, wherein the polypeptide isa synthetic polypeptide.